Anti-inflammatory factor, method of isolation, and use

ABSTRACT

The invention relates to an anti-inflammatory factor isolated from milk, methods of purifying the anti-inflammatory factor resulting in substantially or highly purified preparations and to methods for using this factor to remove adhered neutrophils from endothelial cells, to prevent the emigration of cells from the vasculature and to suppress the response of lymphocytes to foreign antigens.

This application is a continuation-in-part of U.S. patent applicationSer. No. 07/966,741, filed Oct. 27, 1992, now U.S. Pat. No. 5,352,462,which is a continuation-in-part of U.S. patent application Ser. No.07/580,382, filed Sep. 11, 1990, now U.S. Pat. No. 5,242,691, which is acontinuation-in-part of U.S. Ser. No. 177,223, filed Apr. 4, 1988 (nowU.S. Pat. No. 4,956,349) which is a continuation-in-part of U.S. Ser.No. 001,848 filed Jan. 9, 1987 (now U.S. Pat. No. 4,897,265) which is acontinuation-in-part of U.S. Ser. No. 384,625, filed Jun. 3, 1982 (nowabandoned) and a division of U.S. Ser. No. 546,162 filed Oct. 27, 1983(now U.S. Pat. No. 4,636,384) and of U.S. Ser. No. 910,297, filed Sep.17, 1986 (now U.S. Pat. No. 4,919,929) which is a file wrappercontinuation of U.S. Ser. No. 576,001, filed Feb. 1, 1983 (nowabandoned), all of which are fully incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to an anti-inflammatory factor, processesfor its production in substantially pure or highly pure form, and amethod for its use in the treatment of inflammation.

2. Description of the Background Art

Inflammation, as defined in Dorland's Medical Dictionary, is "alocalized protective response elicited by injury or destruction oftissues which serves to destroy, dilute or wall off both the injuriousagent and the injured tissue." It is characterized by fenestration ofthe microvasculature, leakages of the elements of blood into theinterstitial spaces, and migration of leukocytes into the inflamedtissue. On a macroscopic level, this is usually accompanied by thefamiliar clinical signs of erythema, edema, tenderness (hyperalgesia),and pain. During this complex response, chemical mediators such ashistamine, 5-hydroxytryptamine, various chemotactic factors, bradykinin,leukotrienes, and prostaglandins are liberated locally. Phagocytic cellsmigrate into the area, and cellular lysosomal membranes may be ruptured,releasing lytic enzymes. All of these events may contribute to theinflammatory response.

Inflammation in patients with rheumatoid arthritis probably involves thecombination of an antigen (gamma globulin) with an antibody (rheumatoidfactor) and complement causing the local release of chemotactic factorsthat attract leukocytes. The leukocytes phagocytose the complexes ofantigen-antibody and complement and also release the many enzymescontained in their lysosomes. These lysosomal enzymes then cause injuryto cartilage and other tissues, and this furthers the degree ofinflammation. Cell-mediated immune reactions may also be involved.Prostaglandins are also released during this process.

Prostaglandins, which are likely to be generated in inflammation, causeerythema and increase local blood flow. Two important vascular effectsof prostaglandins are not generally shared by other mediators ofinflammation--a long-lasting vasodilator action and a capacity tocounteract the vasoconstrictor effects of substances such asnorepinephrine and angiotensin.

A number of mediators of inflammation increase vascular permeability(leakage) in the post-capillary and collecting venules. In addition,migration of leukocytes into an inflamed area is an important aspect ofthe inflammatory process.

The Arthus reaction is an inflammatory response brought about by theformation of immune complexes at subcutaneous sites where an antigertcomplexes with antibody to that antigen Neutrophils characteristicallyattach to the Fc portion of the immunoglobulin complex that forms at thesubcutaneous injection site where they release digestive enzymes,causing visible acute inflammation. Thus the reaction is primarilyneutrophil-mediated and agents that effect the development of thereaction do so via an effect on these cells.

There are several pathways whereby an agent might interfere withneutrophil migration from the blood vessels to an inflammatory site. Onelikely pathway is the inhibition of margination, the reversible"sticking" of inflammatory cells to the endothelial cell lining of theblood vessel wall. In the normal state about 50% of neutrophils arereversibly adhered, but during an acute inflammatory response, adhesionbecomes much stronger and is a key step in the process of neutrophilmigration. While prostaglandins are unlikely to be directly involved inthe chemotactic response, another product of the metabolism ofarachidonic acid, leukotriene, is a very potent chemotactic substance.

The anti-inflammatory response is any response characterized byintimation as defined above. It is well known to those skilled in themedical arts that the inflammatory response causes much of the physicaldiscomfort, i.e., pain and loss of function, that has come to beassociated with different diseases and injuries. Accordingly, it is acommon medical practice to administer pharmacological agents which havethe effect of neutralizing the inflammatory response. Agents havingthese properties are classified as anti-inflammatory drugs.Anti-inflammatory drugs are used for the treatment of a wide spectrum ofdisorders, and the same drugs are often used to treat differentdiseases. Treatment with anti-inflammatory drugs is not for the disease,but most often for the symptom, i.e., inflammation.

The anti-inflammatory, analgesic, and anti-pyretic drugs are aheterogeneous group of compounds, often chemically unrelated, whichnevertheless share certain therapeutic actions and side-effects.Corticosteroids represent the most widely used class of compounds forthe treatment of the anti-inflammatory response. Proteolytic enzymesrepresent another class of compounds which are thought to haveanti-inflammatory effects. Hormones which directly or indirectly causethe adrenal cortex to produce and secrete steroids represent anotherclass of anti-inflammatory compounds. A number of non-hormonalanti-inflammatory gents have been described. Among these, the mostwidely used are the salicylates. Acetylsalicylic acid, or aspirin, isthe most widely prescribed analgesic-antipyretic and anti-inflammatoryagent. Examples of steroidal and non-steroidal anti-inflammatory agentsare listed in the Physician∝s Desk Reference, 1987 (see pp. 207 and 208for an index of such preparations).

The natural and synthetic corticosteroid preparations cause a number ofsevere side effects, including elevation of blood pressure, salt andwater retention, and increased potassium and calcium excretion.Moreover, corticosteroids may mask the signs of infection and enhancedissemination of infectious microorganisms. These hormones are notconsidered safe for use in pregnant women, and long-term corticosteroidtreatment has been associated with gastric hyperactivity and/or pepticulcers. Treatment with these compounds may also aggravate diabetesmellitus, requiring higher doses of insulin, and may produce psychoticdisorders. Hormonal anti-inflammatory agents which indirectly increasethe production of endogenous corticosteroids have the same potential foradverse side-effects.

The non-hormonal anti-inflammatory agents are synthetic biochemicalcompounds which can be toxic at high doses with a wide spectrum ofundesirable side-effects. For example, salicylates contribute to theserious acid-base balance disturbances that characterize poisoning bythis class of compounds. Salicylates stimulate respiration directly andindirectly. Toxic doses of salicylates cause central respiratoryparalysis as well as circulatory collapse secondary to vasomotordepression. The ingestion of salicylate may result in epigastricdistress, nausea, and vomiting. Salicylate-induced gastric bleeding iswell known. Salicylates can produce hepatic injury, and lead to aprolongation of clotting time. Therefore, aspirin should be avoided inpatients with severe hepatic damage, hypoprothrombinemia, vitamin Kdeficiency, or hemophilia, because the inhibition of platelet hemostasisby salicylates can result in hemorrhage. Salicylate intoxication iscommon, and over 10,000 cases of serious salicylate intoxication areseen in the United States every year, some of them being fatal, and manyoccurring in children. See Goodman and Gilman's The PharmacologicalBasis of Therapeutics, 7th Ed., 1985. Accordingly, in spite of the largenumber of anti-inflammatory agents that are currently available, therestill exists a need for a safe, effective anti-inflammatory productwhich is free of side-effects and adverse reactions.

If a natural food product, such as one derived from milk, for example,could be obtained having anti-inflammatory effects, it would be aneasily administrable, readily available, safe therapeutic composition.

It has been known in the prior art to produce milks having a variety oftherapeutic effects. Beck, for example, has disclosed a milk containingantibody to Streptococcus mutans that has dental caries inhibitingeffect (U.S. Pat. No. 4,324,782). The milk is obtained by immunizing acow with S. mutans antigen in two stages and obtaining the therapeuticmilk therefrom.

Stolle et al. have disclosed a method for treating vascular disorders orpulmonary disorders associated with smoking in an animal which comprisesadministering to the animal milk collected from a cow being maintainedin a hyperimmune state (U.S. Pat. No. 4,636,384). Beck has disclosed amethod for treating intimation in an animal which comprisesadministering to the animal an anti-inflammatory effective amount ofmilk collected from a cow maintained in an anti-inflammatory factorproducing state (U.S. Pat. No. 4,284,623). Heinbach, U.S. Pat. No.3,128,230, has described milk containing globulins of alpha, beta, andgamma components by inoculating a cow with antigenic mixtures. Petersonet al. (U.S. Pat. No. 3,376,198), Holm (U.S. application (published)Ser. No. 628,987), Tunnah et al. (British Patent No. 1,211,876) andBiokema S. A. (British Patent 1,442,283) have also describedantibody-containing milks.

None of the aforementioned references, however, disclose the identity ofthe component or components of therapeutic milks which produce thedesired therapeutic effects. For example, in Beck, U.S. Pat. No.4,284,623, the milk products used as a therapeutic means consist ofeither fluid whole milk, fluid fat-free whey, or whole milk powders.Although each of these milk products has anti-inflammatory properties,the factor or factors that actually provide the therapeutic benefitshave not yet been isolated or identified or purified to homogeneity.

A particular difficulty in obtaining highly purified preparations ofmilk anti-inflammatory factor(s) (MAIF) is the inability to removetightly bound salts from the MAIF by currently used purificationprocedures. One of the problems that this invention adresses, interalia, is the large scale preparation of MAIF including the eliminationof tightly bound salts from the MAIF preparation, thereby resulting inhighly purified MAIF. Further, problems have previously arisen inobtaining highly pure, preparative scale preparations of MAIF whenbeginning with large volumes of starting materials (e.g. 90 liters ofskim milk). The invention provides solutions to these problems.

SUMMARY OF THE INVENTION

The present invention is directed to an anti-inflammatory factor presentin milk and various methods involving the use of the anti-inflammatorypresent in milk. Specifically, the invention is directed to ananti-inflammatory factor produced from milk by removing the fat from themilk; filtering the milk so as to remove molecules with molecularweights greater than about 10,000 daltons; fractionating the filtratecontaining small molecular weight molecules by ion-exchange; furtherenriching ion-exchange fractions in the factor by gel filtration andfurther enriching gel filtration fractions by affinity chromatographyusing a chromatography medium with an affinity for coplanar adjacent cishydroxyl groups.

The invention is further directed to methods for additional purificationof the milk anti-inflammatory factor using, inter alia, HPLC sizeexclusion chromatography and organic partition extraction protocols.

The invention is further directed to methods for using a milkanti-inflammatory factor to prevent neutrophils from adhering to theendothelium of venules or to detach neutrophils which have alreadyadhered to the endothelial cells lining the walls of venules. In thisway, the factor is used to reduce the tissue damage associated with theinflammatory response.

The invention is also directed to a method for using the milkanti-inflammatory factor to prevent interactions between CD18cell-surface antigens and other molecules. It is known that suchinteractions are necessary for the exit of cells from the vasculatureand that such emigration leads to increased tissue damage in animalsduring the inflammatory response. CD18 antigens are also known to beimportant in the immunological response of a host organism to foreignantigens.

Also encompassed by the invention is the use of the anti-inflammatoryfactor in mammals to prevent the emigration of cells from thevasculature and to suppress the mitogenic response of lymphocytes toforeign antigens.

BRIEF DESCRIPTION OF THE DRAWINGS

A more complete appreciation of the invention and many of the attendantadvantages thereof will be readily obtained as the same becomes betterunderstood by reference to the following detailed description whenconsidered in connection with the accompanying drawings, wherein:

FIG. 1. Isolation of the anti-inflammatory factor by ion-exchangechromatography on a column of DEAE-cellulose.

FIG. 2. Fractionation of the anti-inflammatory factor containing peak(second) from DEAE-cellulose chromatography (FIG. 1) on a Sephadex G-10molecular sieve column.

FIG. 3. Effect of immune milk on carrageenan-induced edema in rats (pawweight, % control paw, mean±sem, n=10).

FIG. 4. Effect of intraperitoneal administration of theanti-inflammatory factor on footpad edema in rats (μL, mean±SD, n=6).

FIG. 5. Intraperitoneal dose-response curve for the anti-inflammatoryfactor in rat paw edema test (% control, mean±SD, n=6).

FIG. 6. Effect of hyperimmune milk factor vs. placebo (lactose) onfootpad edema in rats (% control, mean±SD, n=6).

FIG. 7. Effect of iv and oral MAIF on footpad edema in rats (% control,mean±SD, n=6).

FIG. 8. Effect of low iv dosage of MAIF on footpad edema in rats (%control, mean±SD, n=6).

FIG. 9. Intravenous dose-response curve for MAIF in rat paw edema test(% control, mean±SD, n=6).

FIG. 10. Run 1, twin herd/ultrafiltration experiments (% average controledema, mean±SD, n=6).

FIG. 11. Run 2, twin herd/ultrafiltration experiments (% average controledema, mean±SD, n=6).

FIG. 12. Run 3, twin herd/ultrafiltration experiments (% average controledema, mean±SD, n=6).

FIG. 13. Effect of various treatments of MAIF on inhibition of footpadedema in rats (μL footpad edema, mean±SD, n=6).

FIG. 14. Effect of fractions of MAIF and of immune wpc on inhibition offootpad edema in rats (μL footpad edema, mean±SD, n=6).

FIG. 15. Effect of five different anesthetics on the response tocarrageenan in the rat footpad. The accumulation of edema was monitoredat selected intervals in the same animals. n=6 for each data point.

FIG. 16. Demonstration of the biphasic nature of the response tocarrageenan in the rat footpad. n=5 for each data point. Ether was usedas the anesthetic.

FIG. 17(A-B). MAIF, administered at either 5 mg per rat (A) or 40 mg perrat (B) does not inhibit the inflammatory response to carrageenan inether-anesthetized rats. n=4 for all data points.

FIG. 18. Suppression of carrageenan-induced edema accumulation duringthe secondary, phagocytic-cell mediated, response by 40 mg of MAIFinjected i.v. at the time of carrageenan challenge (time 0). n=12 foreach data point in the control group and n=10 for each data point in theMAIF-treated group.

FIG. 19. Effect of MAIF, given i.v. at 4 mg per rat at different times,on the response to carrageenan in the rat footpad. Edema was assessed 4hours after challenge in all cases. n=12 for each data point.

FIG. 20. Effect of 20 mg of MAIF injected i.v. on the reverse passiveArthus reaction. *=p<0.01; **=p<0.05.

FIG. 21. Effect of decreasing doses of MAIF on the ability ofneutrophils to emigrate from the vasculature into subcutaneouslyimplanted sterile sponges. *=p<0.01.

FIG. 22. Effect of MAIF, administered at a dose of 20 mg per rat, toinhibit the ability of inflammatory cells to accumulate insubcutaneously implanted sponges when administered at the time ofimplant or up to 120 minutes after implant. *=p<0.01.

FIG. 23. Time course of the cellular inflammatory infiltration intosubcutaneously implanted sponges in normal animals.

FIG. 24. Effect of preparations of anti-inflammatory factor onplatelet-activating factor (PAF) induced adhesion of neutrophils tovenules.

FIG. 25. Effect of preparations of anti-inflammatory factor onPAF-induced neutrophil emigration.

FIG. 26. Effect of preparations of anti-inflammatory factor onPAF-induced flux of neutrophils through venules.

FIG. 27. Reversal of neutrophil adhesion by preparations ofanti-inflammatory factor. 27a shows the effect of the MAIF preparation(40 mg/rat) in reducing the number of neutrophils adhering to venules inresponse to PAF. 27b shows the effect of the MAIF preparation (40mg/rat) on new neutrophil-endothelial cell adhesions.

FIG. 28. Effect of preparations of anti-inflammatory factor on thevelocity of neutrophils in venules.

FIG. 29. Effect of preparations of anti-inflammatory factor on thevelocity of red blood cells in venules.

FIG. 30. Effect of anti-inflammatory factor on leukocyte flux invenules.

FIG. 31. Effect of 40 mg of the MAIF preparation administered i.v. onthe number of circulating neutrophils and lymphocytes in the 24 hoursfollowing injection.

FIG. 32. Dose-response relationship between i.v. administration of theMAIF preparation and circulating leukocyte numbers (p<0.01).

FIG. 33(A-E). Effect of anti-inflammatory factor on various aspects oflymphocyte function. 33a shows the effect of prior administration offactor on the response of host T lymphocytes to foreignhistocompatibility antigens. 33b shows the results obtained whenlymphocytes from MAIF treated rats are injected into untreated rats. 33Cand 33D show the effect of MAIF treatment on spleen weight and spleencell amber in rats. 33E shows the effect of MAIF treatment on theconcanavalin A stimulated mitogenic response of lymphocytes.

FIG. 34. Suppression of infection-induced edema by 40 mg of MAIFinjected i.v. The mean values of the two groups were: controls, 87±22μL; MAIF, 45±17 μL; p<0.01.

FIG. 35. Effect of MAIF given i.v. at 40 mg per rat on bacterialreplication and subcutaneously implanted, E. coli-infected sponges.

FIG. 36(A-B). Inhibition of inflammatory cell infiltration into infectedsponges by MAIF (40 mg per rat, i.v.).

FIG. 37. Effect of MAIF (40 mg per rat, i.v.) on suppression of theintermediate phase (4-16 hours) of inflammatory fluid accumulation in E.coil-infected sponges.

FIG. 38. Effect of 40 mg of MAIF, given intravenously at the time ofchallenge and 48 hours later, on the pathogenesis of experimentalpyelonephritis. The dotted line on the left-hand graph represents themean background kidney weight. *=p<0.01; **=p<0.02.

FIG. 39. Comparison of standardized hyperimmune and control MAIF. MAIFprepared by standardized methods was tested at doses of 0.5, 1.5, 3, 5and 8 mg/120-150 gm. female rat for their ability to inhibit themigration of neutrophils to inflammatory sites. Reference to"commercial" is to off the shelf powdered skim milk.

FIG. 40. Comparison of MAIF and other milk components. The activity ofstandardized MAIF prepared from hyperimmune milk was compared withsialic acid and orotic acid, known components of milk believed to haveanti-inflammatory activity.

FIG. 41. Comparison of standardized MAIF from hyperimmune milk andanti-inflammatory drugs (Indomethacin and aspirin).

FIG. 42. Analysis of the composition of the DEAE-derived MAIF by sizeexclusion HPLC.

FIG. 43. Analysis of the dried ethyl acetate fraction in the neutrophilmigration inhibition assay demonstrated strong MAIF activity.

FIG. 44(A-B). Analysis of the MAIF compound on an aminopropyl weak anionexchange column before and after extraction. "Before" (FIG. 44A) and"after" (FIG. 44B) refers to preparations before and after organicpartition chromatography. Note the significant disappearance of shoulder"A" in the shifted elution pattern of FIG. 44B.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The invention comprises the isolation and purification of ananti-inflammatory factor from milk and the administration of said factorto an animal for the purpose of treating anti-inflammatory disorders.Except as otherwise indicted, the following definitions apply:

By the term "milk anti-inflammatory factor" is intended a factorobtained from either hyperimmune milk or normal cow's milk. By the term"substantially pure milk anti-inflammatory factor" is intended, for thepurpose of this invention, an anti-inflammatory factor that elutes as asingle major symmetrical peak on HPLC chromatography, after removal ofhigh molecular weight substances (>10,000 daltons) and isolation of thelow molecular weight, negatively-charged species by ion-exchangechromatography. By the term "highly purified" or "highly pure" isintended an anti-imflamatory factor which has an elution patternsimilar, though not necessarily identical to that of FIG. 44B, onaminopropyl weak anion exchange column. A particular characteristic ofsuch an elution profile is significant loss of shoulder A (as seen inFIG. 44A) as seen in the shifted profile of FIG. 44B. Both normal milkand hyperimmune milk can be processed by the methods described herein toobtain the anti-inflammatory factor.

By the term "hyperimmune milk" is intended, for the purpose of thisinvention, milk obtained from milk-producing animals maintained in ahyperimmune state, the details for hyperimmunization being described ingreater detail below.

By the term "whey" is intended, for the purpose of this invention, milkfrom which cream has been removed.

By the term "normal milk" is intended for the purpose of the inventionmilk that is obtained from milk-producing animals by conventional meansand dairy practices.

By the term "milk-producing animal" is intended, for the purpose of thisinvention, mammals that produce milk in commercially feasiblequantities, preferably cows, sheep and goats, more preferably dairy cowsof the genus Bos (bovid), particularly those breeds giving the highestyields of milk, such as Holstein.

By the term "bacterial antigen" is intended, for the purpose of thisinvention, a lyophilized preparation of heat-killed bacterial cells.

By the term "microencapsulated form" is intended, for the purpose ofthis invention, polymeric microparticles encapsulating one or morebacterial antigens for administration to milk-producing animals.

By the term "inflammation" is intended, for the purpose of thisinvention, a localized protective response elicited by injury ordestruction of tissues which serves to destroy, dilute or wall off boththe injurious agent and the injured tissue, characterized in the acuteform by the classical sequence of pain, heat, redness, swelling, andloss of function, and histologically involving a complex series ofevents, including dilatation of the arterioles, capillaries, and venuleswith increased permeability and blood flow, exudation of fluidsincluding plasma proteins, and leukocyte migration into the inflammatoryfocus.

By the term "treating" is intended, for the purposes of this invention,that the symptoms of the disorder and/or pathogenic origin of thedisorder be ameliorated or completely eliminated.

By the term "administer" is intended, for the purpose of this invention,any method of treating a subject with a substance, such as orally,intranasally, parenterally (intravenously, intramuscularly, orsubcutaneously), or rectally. By the term "animal" is intended, for thepurpose of this invention, any living creature that is subject toinflammation, including humans, farm animals, domestic animals, orzoological garden animals.

Examples of inflammatory conditions that may be treated by the isolatedand purified milk product of the present invention are conditionsselected from the group consisting of acute and subacute bursitis, acutenon-specific tendinitis, systemic lupus erythematosus, systemicdermatomyositis, acute rheumatic carditis, pemphigus, bullousdermatitis, herpeteformis, severe erythema, multiform exfoliativedermatitis, cirrhosis, seasonal perennial rhinitis, bronchial asthma,ectopic dermatitis, serum sickness, keratitis, opthalmicus iritis,diffuse ureitis, chorditis, optic neuritis, sympathetic ophthalmia,symptomatic sarcoidosis, Loeffler's syndrome, berylliosis, hemolyticanemia, mastitis, mastoiditis, contact dermatitis, allergicconjunctivitis, psoriatic arthritis, ankylosing spondylitis, acute goutyarthritis, and herpes zoster. Further, the isolated and purified milkproduct may be used to treat individuals who are exposed to potentiallyinflammatory agents.

The invention is based in part on the discovery that when amilk-producing animal such as a bovid is brought to a specific state ofhyperimmunization, the animal will produce milk which has supranormallevels of the highly beneficial anti-inflammatory factor, said factornot only suppressing the symptoms of inflammation in man and otheranimals, but also being a prophylactic agent in anticipation of thepresence of inflammatory agents in the recipient. By the term"supranormal levels" is intended levels in excess of that found in milkfrom non-hyperimmunized animals. The induction of immune sensitivityalone is insufficient to cause the appearance of supranormal levels ofMAIF in milk, as is shown by the fact that normal cow's milk does notcontain these supranormal levels, even though the cows have becomesensitized against various antigens during normal immunization againstcow diseases and during normal exposure to the environment. It is onlyin specific hyperimmune states that the milk has the desired supranormallevels.

This special state may be achieved by administering an initialimmunization, followed by periodic boosters with sufficiently high dosesof specific antigens. The preferred dosage of booster should be equal toor greater than 50% of the dosage necessary to produce primaryimmunization of the bovid. Thus, there is a threshold booster dosagebelow which the properties are not produced in the milk, even though thecow is in what normally would be called an immune state. In order toachieve the requisite hyperimmune state, it is essential to test thehyperimmune milk after a first series of booster administrations. If thebeneficial factors are not present in the milk, additional boosters ofhigh dosage are administered until the properties appear in the milk.

The process of producing the hyperimmune milk containing supranormallevels of anti-inflammatory factor is disclosed in co-pending U.S.patent application Ser. No. 580,382, filed Sep. 11, 1990 and also inU.S. Ser. No. 355,786, filed May 22, 1989 (now U.S. Pat. No. 5,106,618,a file wrapper continuation of U.S. Ser. No. 069,139, filed Jul. 2,1987) and in U.S. Ser. No. 910,297, filed Sep. 17, 1986 (now U.S. Pat.No. 4,919,929, a file wrapper continuation of U.S. Ser. No. 576,001,filed Feb. 1, 1983), all of which are incorporated herein by referencein their entirety. In summary, one process of producing the hyperimmunemilk containing supranormal levels of anti-inflammatory factor comprisesthe following steps: (1) antigen selection; (2) primary immunization ofthe bovid; (3) testing the serum to confirm sensitivity induction; (4)hyperimmunization with boosters of appropriate dosage; and, optionally,(5) testing the milk for anti-inflammatory properties; (6) collectingthe milk from the hyperimmune bovid; and (7) processing the milk toisolate the MAIF.

Step 1: Any antigens or combination of antigens may be employed. Theantigens can be bacterial, viral, protozoan, fungal, cellular, or anyother substances to which the immune system of a milk-producing animalwill respond. The critical point in this step is that the antigen(s)must be capable, not only of inducing immune and hyperimmune states inthe milk-producing animal, but also of producing supranormal levels ofanti-inflammatory factor in the milk. Any antigen can be used to producesupranormal levels of factor. One preferred vaccine is a mixture ofpolyvalent bacterial antigens, referred to as Series 100 vaccine,described in detail in Example 1A below.

Step 2: The antigen(s) can be administered in any method that causessensitization. In one method, a vaccine composed of antigen derived from1×10⁶ to 1×10²⁰, preferably 10⁸ to 10¹⁰, most preferably 2×10⁸,heat-killed bacteria is administered by intramuscular injection.However, other methods such as intravenous injection, intraperitonealinjection, rectal suppository, or oral administration may be used.

Step 3: It is necessary to determine whether or not the milk-producinganimal has become sensitive to the antigen There are a number of methodsknown to those skilled in the art of immunology to test for sensitivity(Methods in Immunology and Immunochemistry, William, C. A., and Chase,W. M., Academic Press, New York, vols. 1-5 (1975)). The preferred methodis to use a polyvalent vaccine comprising multiple bacterial species asthe antigen and to test for the presence of agglutinating antibodies inthe serum of the animal before and after challenge with the vaccine. Theappearance of milk antibodies after immunization with the vaccineindicates sensitivity; at this point it is possible to proceed to step4.

Step 4: This involves the induction and maintenance of the hyperimmunestate in the sensitized animal. This is accomplished by repeated boosteradministration at fixed time intervals of the same polyvalent vaccinethat was used to achieve the primary sensitization. A two-week boosterinterval is optimal for polyvalent bacterial antigens. However, it isnecessary to ensure that the animal does not pass from a hyperimmunestate to a state of immune tolerance to the antigen.

In a preferred embodiment, hyperimmunization of bovids may be achievedby a single administration of microencapsulated vaccine, prepared asdescribed in detail in Example 1B below. The advantage of the controlledrelease form of hyperimmunization is that the constant exposure to theantigen ensures that the animal remains in the hyperimmune state.

In an alternative embodiment, it is also possible to combine differentimmunization procedures, e.g., simultaneously administeringmicroencapsulated and liquid antigen, or intramuscular injection forprimary immunization, and booster doses by oral administration orparenteral administration by microencapsulation means. Many differentcombinations of primary and hyperimmunization are known to those skilledin the art.

Step 5: It is necessary to test the milk for anti-inflammatory activitylevels. This can be accomplished by any research technique that teststhe effects of either the hyperimmune milk or products derived therefromupon inflammation. Chemical-induced inflammation of the rat paw is astandard assay for anti-inflammatory drugs.

Step 6: This involves the collection and processing of the milk. Themilk can be collected by conventional methods. Processing the milk toisolate the anti-inflammatory factor is described below.

The simplest process for isolating, purifying and testing theanti-inflammatory factor comprises the following steps:

1. defatting the hyperimmune milk to produce skim milk;

2. removing casein from skim milk to produce whey;

3. removal from the whey macromolecules of molecular weight greater thanabout 10,000 daltons by ultrafiltration;

4. fractionating the product from step 3 using an ion-exchange resincolumn to isolate a negatively-charged anti-inflammatory species ofmolecular weight less than about 10,000 daltons;

5. separating the negatively-charged species from step 4 by molecularsieve chromatography; and

6. biological assay of the anti-inflammatory factor preparation fromstep 5.

In an alternative preferred embodiment, the fractions from molecularsieve chromatography that have biological activity are further purifiedby filtration through a membrane that retains macromolecules ofmolecular weight greater than about 5000 daltons.

Another preferred embodiment further comprises additional purificationof the MAIF by HPLC size exclusion chromatography and organic partitionextraction.

7. The anti-inflammatory action of the milk factor is tested on edemathat is caused by the injection of a solution of carrageenan into thepaw of rats. The rat paw test is the standard animal test foranti-inflammatory drugs. Winter, C. A., Risley, G. A., Nuss, A. W.,"Carrageenan-Induced Edema in the Hind Paw of the Rat as an Assay forAnti-inflammatory Drugs," Proc. Soc. Exper. Biol. Med. 3:544 (1967).Alternatively, one can use a pleural neutrophil migration inhibitionasssay as described in Example 24. Vinegar et al., "Some QuantitativeCharacteristics of Carrageenan-induced Pleurisy in the Rat," Proc. Soc.Exp. Biol. Med. 143:711-714 (1973); Arnmendola, G. et al. "LeukocyteMigration and Lysozomal Enzymes Release in Rat Carrageenan Pleurisy,"Agents and Actions 5:250-255 (1975); Vinegar, R. et al. "QuantitativeStudies of the Pathway to Acute Carrageenan Inflammation.". Fed. Proc.35:2447-2456 (1976). A variety of other tests may be used. Wetnick, A.S., and Sabin, C., "The Effects of Clonixin and Bethaurethasone onAdjuvant-Induced Arthritis and Experimental Allergic Encephalomyelitisin Rats," Jap. J. Pharm. 22:741 (1972). However, the rat paw test is themost simple and direct test available, and has been shown to besatisfactory for all anti-inflammatory drugs. This test has beendescribed in detail in Beck, U.S. Pat. No. 4,284,623, which isincorporated herein by reference to the extent that it describes the ratpaw test. Briefly, the test involves the injection of a small quantityof carrageenan into the footpad of adult white rats. This is known toinduce an inflammatory response. The resulting degree of swelling can bequantified. Samples containing an anti-inflammatory factor areadministered to the rat by a suitable route, preferably byintraperitoneal injection, and the blockade or amelioration of theinflammatory process quantified by either volumetric or gravimetricmethods.

In sugary, one can isolate the anti-inflammatory factor fromhyperimmunized milk by following a process of defatting the milk,removing casein, removing macromolecules of greater than 10,000 daltons,and continuing with ion exchange and molecular sieve chromatography. Thebiological activity of appropriate preparations of anti-inflammatoryfactor can be tested by doing a dose-response experiment on rats asdescribed herein.

In an additional preferred embodiment of the present invention, theanti-inflammatory factor present in hyperimmunized milk is purifiedusing a combination of steps involving: filtration on a membrane capableof separating molecules based upon their molecular weights; ion-exchangechromatography; molecular sieve chromatography; and affinitychromatography (Example 15). The preferred first step comprisesfiltering hyperimmune skim milk, produced as described above, through amembrane which retains molecules with molecular weights of about 10,000daltons or more. The material passed by the membrane (i.e. the filtrateor permeate) is collected and used in further purification steps.Devices and membranes for performing such filtrations are well-known inthe art.

The preferred step following filtration is ion-exchange chromatographyon a anion exchanger. Exchangers having diethylaminoethyl groups havebeen found to effectuate good separations but it is expected that otheranion exchangers could be used as well. It is preferred that the solidsupport of the ion-exchanger be capable of maintaining high flow rates.Sepharose has been found to be suitable for this purpose.

The preferred step after ion-exchange chromatography is gel filtrationchromatography. A column packing for this step should be chosen which iscapable of fractionating molecules with molecular weights of less than10,000 daltons. The preferred packing is Toyopearl HW-40 (Rohm and Haas)but other packings well known in the art could be used as well. Examplesof other packings that could be used and which are commerciallyavailable are polymeric carbohydrate based packings, e.g. Sephadex G-10or G-25 (Pharmacia), or polyacrylamide based packings, e.g. Biogel P-2,P-4, P-6, P-10 or P-30, (Bio-Rad).

The preferred step after gel filtration chromatography is affinitychromatography on a boronate affinity support. These supports have beenfound to be effective at fractionating low molecular weight compoundswith cis-diol groups. The preferred support is AffiGel 601 (Bio-Rad).This is a boronate derivative of the polyacrylamide gel filtrationsupport gio-Gel P-6 (also sold by Bio-Rad).

The preferred mode of storage for preparations after the ion exchange,gel filtration or affinity chromatography steps is as a lyophilizedpowder. The filtrate collected in the first purification step may bestored refrigerated until use. The activity of the anti-inflammatoryfactor resulting from the purification may be determined using the ratpaw test described above.

In another preferred embodiment of the present invention, theanti-inflammatory factor present in hyperimmunized milk is analyticallyor preparative purified using a combination of steps involving:ultrafiltrationion with a membrane having a molecular weight cutoff of10,000 daltons, ion exchange chromatography, size exclusionchromatography on a preparative HPLC column and organic partitionextraction.

In a preferred first step, a large volume (e.g. 90 liter of skimmed milkis passed through an ultrafiltration membrane with a 10,000 daltoncutoff. The resulting permeate (<10K permeate) is collected for furtherpurification steps.

The preferred step following the above ultrafiltration is applying alarge volume (e.g. 60 liters) of permeate from the previous step to anion exchange column. The eluate can then be lyophilized.Diethyl-aminoethyl (DEAE)-Sepharose Fast Flow exchange resin has beenfound to effectuate good separation.

The preferred step after the above ion-exchange chromatography isplacing on a peparative HPLC column a large quantity (e.g. 100 mg.) ofthe purified preparation from the ion-exchange column. Even largerquantities can be obtained by separating multiple samples of 100 mg eachon the HPLC column and collecting them into the same set of tubes. Thetubes can then be pooled and their contents lyophilized.

The next preferred step is organic partition extraction which entailstaking 50-100 mg. of the lyophilized HPLC column samples and extractingwith n-hexane to remove neutral lipids, acidification and thenreextraction with ethyl acetate. The analysis of a preparation on anaminopropyl weak anion exchange column before and after organicpartition extraction is shown in FIG. 44.

Results of experiments described in Example 16 indicate thatpretreatment of animals with preparations of anti-inflammatory factorreduces the platelet activating factor (PAF) stimulated adhesion ofneutrophils to the endothelial cells which line venules and reduces therate at which neutrophils emigrate from venules. In addition, theadministration of preparations of the factor after treatment of animalswith PAF was found to reduce the number of neutrophils adhering toendothelial cells. To the extent that patients or animals may benefitfrom these effects, the present invention encompasses the use ofpreparations of the anti-inflammatory factor. This is true regardless ofthe particular disease involved. Similarly, the data in Example 16indicates that the anti-inflammatory factor causes its effects onadhesion and emigration by interacting directly with cell-surface CD18antigens and preventing other molecules from interacting with thisglycoprotein complex. The present invention encompasses the use ofpreparations of the anti-inflammatory factor for this purpose as well.

A shown in Example 18, the administration of a preparation ofanti-inflammatory factor to animals suppresses the Host vs. Graft butnot the Graft vs. Host reaction and causes an increase in spleen weightand in the number of splenic lymphocytes. The lymphocyte response toConcanavalin A was also found to be abrogated by the preparation. Thesedata indicate that the anti-inflammatory factor is useful in theinhibition of tissue destructive infectious processes, and in situationswhere suppression of lymphocyte function is desirable.

As shown in FIG. 43, desalting of the MAIF (obtained by organic paritionextraction) results in dramatic increases in purification as indicatedby as much as a 10,000 fold lower dose of MAIF being required to obtainsimiliar levels of migration inhibition of rat pleural leukocytes.

The compositions of the present invention may be administered by anymeans that provide anti-inflammatory activity. For example,administration may be parenteral, subcutaneous, intravenous,intramuscular, intraperitoneal or oral.

Solid dosage forms for oral administration include capsules, tablets,pills, powders and granules. In such solid dosage forms the activecompound is admixed with at least one inert diluent such as sucrose,lactose or starch. Such dosage forms can also comprise, as is normalpractice, additional substances other than inert diluent. In the case ofcapsules, tablets, and pills, the dosage forms may also comprisebuffering agents. Tablets and pills can additionally be prepared with anenteric coating.

Liquid dosage forms for oral administration include pharmaceuticallyacceptable emulsion, solutions, suspensions, syrups and elixirscontaining inert diluents commonly used in the pharmaceutical art.Besides inert diluents, such compositions can also include adjuvants,such as wetting agents, emulsifying and suspending agents, andsweetening.

Preparations according to this invention for parenteral administrationinclude sterile aqueous or nonaqueous solutions, suspensions oremulsions. Examples of nonaqueous solvents or vehicles are propyleneglycol, polyethylene glycol, vegetable oils such as olive oil andinjectable organic esters such as ethyl oleate.

The dosage of active ingredients in the composition of this inventionmay be varied; however it is necessary that the amount of the activeingredient shall be such that a suitable dosage form is obtained. Theselected dosage form depends upon the desired therapeutic effect, on theroute of the administration and on the duration of the treatment.

Administration dosage and frequency will depend on the age and generalhealth condition of the patient, taking into consideration thepossibility of side effects. Administration will also be dependent onconcurrent treatment with other drugs and patients tolerance of theadministered drug.

Having now described the invention in general terms, the same will befurther described by reference to certain specific examples that areprovided herein for purposes of explanation only, and are not intendedto be limiting unless otherwise specified.

EXAMPLE 1A Preparation of S-100 Vaccine

A bacterial culture containing the spectrum of bacteria shown in Table 1below as obtained from the American Type Culture Collection wasreconstituted with 15 ml of media and incubated overnight at 37° C. Oncegood growth was obtained, approximately one-half of the bacterialsuspension was employed to inoculate one liter of broth with theinoculate being incubated at 37° C. The remaining suspension wastransferred to sterile glycol tubes and stored at -20° C. for up to sixmonths.

After good growth was visible in the culture, the bacterial cells wereharvested by centrifugation of the suspension for 20 minutes to removethe media. The bacterial pellet obtained was resuspended in sterilesaline solution and the bacterial sample was centrifuged three times towash the media from the cells. After the third sterile saline wash, thebacterial pellet obtained upon centrifugation was resuspended in a smallamount of double distilled water.

The media-free bacterial suspension was heat-killed by placing thesuspension in a glass flask in an 80° C. water bath overnight. Theviability of the broth culture was tested with a small amount ofheat-killed bacteria. Broth was inoculated with heat-killed bacteria,incubated at 37° C. for five days and checked daily for growth, as thebacteria have to be killed for use in the vaccine.

The heat-killed bacteria were lyophilized until dry. The dry bacteriawere then mixed with sterile saline solution to a concentration of2.2×10⁸ bacterial cells/ml saline (1.0 optical density reading at 660nm).

                  TABLE 1                                                         ______________________________________                                        S-100 Bacteria List                                                                                      Gram                                               Name              Media    + or -   ATTC #                                    ______________________________________                                        1.   Staph. aureus    BHI      +      11631                                   2.   Staph. epidermidis                                                                             BHI      +       155                                    3.   Strep. pyogenes, A. Type 1                                                                     APT      +       8671                                   4.   Strep. pyogenes, A. Type 3                                                                     APT      +      10389                                   5.   Strep. pyogenes, A. Type 5                                                                     APT      +      12347                                   6.   Strep. pyogenes, A. Type 8                                                                     APT      +      12349                                   7.   Strep. pyogenes, A. Type 12                                                                    APT      +      11434                                   8.   Strep. pyogenes, A. Type 14                                                                    APT      +      12972                                   9.   Strep. pyogenes, A. Type 18                                                                    APT      +      12357                                   10.  Strep. pyogenes, A. Type 22                                                                    APT      +      10403                                   11.  Aerobacter aerogenes                                                                           BHI      -       884                                    12.  Escherichia coli BHI      -        26                                    13.  Salmonella enteritidis                                                                         BHI      -      13076                                   14.  Pseudomonas aeruginosa                                                                         BHI      -       7700                                   15.  Klebsiella pneumoniae                                                                          BHI      -       9590                                   16.  Salmonella typhimurium                                                                         BHI      -      13311                                   17.  Haemophilus influenzae                                                                         BHI      -       9333                                   18.  Strep. mitis     APT      +       6249                                   19.  Proteus vulgaris BHI      -      13315                                   20.  Shigella dysenteriae                                                                           BHI      -      11835                                   21.  Diplococcus pneumoniae                                                                         APT      +       6303                                   22.  Propionibacter acnes                                                                           Broth    +      11827                                   23.  Strep. sanguis   APT      +      10556                                   24.  Strep. salivarius                                                                              APT      +      13419                                   25.  Strep. mutans    BHI      +      25175                                   26.  Strep. agalactiae                                                                              APT      +      13813                                   ______________________________________                                    

Cows were given daily injections of 5 ml samples of the polyvalentliquid vaccine. Antibody (IgG) titer levels for the injected cattle weredetermined periodically by using an enzyme-linked immunoassay for bovineantibody against the polyvalent antigen

EXAMPLE 1B Immunization Procedures

Heat-killed bacteria were prepared in the manner described above. Thepolyvalent antigen sample (S-100) obtained was microencapsulated by aconventional phase-separation process to prepare a polyvalentantigen-containing microparticle product. Generally, theantigert-containing shaped matrix materials are formed from polymers ofbiocompatible material, preferably biodegradable or bioerodablematerials, preferably polylactic acid, polyglycolic acid, copolymers oflactic and glycolic acids, polycaptolactone, copolyoxalates, proteinssuch as collagen, fatty acid esters of glycerol, and cellulose esters.These polymers are well known in the art and are described, for example,in U.S. Pat. No. 3,773,919; U.S. Pat. No. 3,887,699; U.S. Pat. No.4,118,470; U.S. Pat. No. 4,076,798; all incorporated by referenceherein. The polymeric matrix material employed was a biodegradablelactide-glycolide copolymer.

Heat-killed bacterial antigens are encapsulated in such matrixmaterials, preferably as microspheres of between 1-500 microns diameter,preferably 10-250 microns. The encapsulation processes are conventionaland comprise phase separation methods, interfacial reactions, andphysical methods. Many combinations of matrices and many concentrationsof assorted antigens may be employed, in order to provide for optimalrates of release of bacterial antigens to the host body from themicroparticles. These combinations can be determined by those skilled inthe art without undue experimentation.

The microparticles in the example were less than 250 microns indiameter. Approximately 750 mg of microparticles containing 22% (16.5mg) of polyvalent antigert was then suspended in about 3 cc of a vehicle(1 wt. % Tween 20 and 2 wt. % carboxymethyl cellulose in water).

A small group of cattle was selected from a larger herd of cattle. Fiveof these randomly selected cattle were selected as controls. Four cattlewere injected intramuscularly with microparticles containing polyvalentantigen Microparticle samples were sterilized with 2.0 mRad of gammaradiation. Antibody (IgG) titer levels were determined periodically fromsamples of cows' milk obtained from the inoculated cows, as well as fromthe control COWS.

EXAMPLE 2 Isolation of MAIF Factor from Hyperimmunized Milk

Step 1: Milk Filtrate Preparation

Twenty liters of fresh milk from hyperimmunized cows were run through acream separator (DeLaval Model 102) to remove the fat.

The resulting sixteen liters of skimmed milk was ultra-filtered toremove the high molecular weight species (over 10,000 chitons) using ahollow fiber diafiltration/concentrator (Amicon DL-10L). Theconcentrator is equipped with two 10,000 daltons molecular weightcut-off cartridges (Amicon H₅ P₁₀₋₄₃). The skimmed milk was run at thepump speed of 80 on the meter and inlet and outlet pressure of 30 psiand 25 respectively.

Twelve liters of the filtrate (<10,000 daltons) coming out of thecartridges at the flow rate of four liters per hour was frozen orlyophilized for storage and for further purification.

Step 2: Ion-Exchange Chromatography

The milk anti-inflammatory factor, in the filtrate was first isolated byan anion exchange chromatography column.

In this procedure, DEAE-Sepharose CL-6B gel (Pharmacia) was used to packa 5×10 cm glass column which was equilibrated with sterile doubledistilled water, pH 7.0.

One liter of filtrate (<10,000) was applied to the column and elutedwith sterile double distilled water, pH 7.0 at the flow rate of 160 mlper hour. Ten milliliter fractions were collected and monitored at 280nm in an LKB Uvicord 4700 absorptiometer with an optical density printedout on a connected recorder (Pharmacia REC-482).

Substances other than the anti-inflammatory factor having positive andneutral charges are not bound to the DEAE-Sepharose gel. They are elutedat the fallthrough peak (first peak). The anti-inflammatory factorcarrying a negative charge is retained by the gel.

To elute the factor, the column was eluted with a stepwise gradientusing sterile physiological saline, pH 7.0. A typical profile is shownin FIG. 1. Bioassay of the individual fractions revealed that the secondpeak contains the factor. Fractions comprising the second peak and itsshoulder are used for further purification. Recovery studies show that8.8 grams of dried powder were obtained by this process.

Step 3: Gel Filtration Chromatography

The second peak obtained from Step 2 contains the anti-inflammatoryfactor and other negatively charged molecules; therefore, an additionalrefining step was needed. To achieve further purification, it isconvenient to use a gel filtration column to separate various componentson the basis of molecular weight.

In this process, Sephadex G-10 resin (Pharmacia) was packed into a2.5×80 cm glass column and equilibrated with sterile double distilledwater, pH 7.0. Two grams of the second fraction from Step 2 wasredissolved in sterile double distilled water and applied to the top ofthe column. The column was eluted at the flow rate of 30 ml per hour.Fractions (3.3 ml) were collected and monitored at 254 nm and 280 nm(Pharmacia Duo Optical Unit) with optical density printed out on aconnected recorder (Pharmacia REC-482).

Typically, there were 3 peaks shown in the elution profile asillustrated in FIG. 2. The first and second peaks containedanti-inflammatory activity.

The first peak is an aggregate that forms on the G-10 column whichcontains the active factor.

The second peak contains the nonaggregated form of the factor. Both theaggregate form (peak 1) and the nonaggregated form (peak 2) arebiologically active in rat bioassay.

EXAMPLE 3 Characterization of Milk Anti-inflammatory Factor

The molecular weight of the non-aggregated form of factor prepared bythe method described above was found to be less than 10,000 daltons.This was deduced from the fact that the first step in the isolation ofthe factor from whey was by ultrafiltration using a membrane that doesnot allow the passage of molecular weight species >10,000 daltons.

The factor has a negative charge. This was determined by applying milkultrafiltrate to a DEAE cellulose ion exchange column. Theanti-inflammatory activity did not elute from the column with water.Changing the elution media to sodium chloride (0.9% pH) caused theelution of several peaks (FIG. 1). Neutral and positive charged speciesdo not adhere to the ion exchange resin, and negative charged speciesare eluted by increasing the salt concentration. When the less than10,000 dalton molecular weight permeate was applied to the DEAE column,neutral salts and sugars eluted with water (Peak 1, FIG. 1). Threedistinct peaks eluted when the buffer was changed to saline (Peaks 24).The second peak and its shoulder contained anti-inflammatory biologicalactivity in the rat assay. It is concluded, therefore, that the factorhas a negative charge.

Mother chemical characteristic of the factor is that it forms anaggregate during the process of removing salt. This property becameapparent when <10,000 dalton molecular weight permeate was passed over aSephadex G-10 column, equilibrated with double distilled water andeluted with water at a pH of 7 (FIG. 2). Three peaks eluted from theG-10 column; the first peak eluted with the void volume suggesting amolecular weight equal to or greater than 10,000 dalton. This wasunexpected because molecules greater than 10,000 daltons had previouslybeen removed from this sample by ultrafiltration. The second peak elutedin the position expected for the anti-inflammatory factor. Both thefirst and second peaks exhibited anti-inflammatory biological activityin the rat paw assay, whereas the third peak lacked activity. It wassurprising to find that both the first and second peaks hadanti-inflammatory biological activity. The material recovered from thefirst peak of the G-10 column (Step 3) was lyophilized and applied to aG-100 column; a single peak was eluted with the void volume, suggestinga molecular weight of 100,000 daltons or greater. The Step 3 G-10 columnremoves salt at the same time it separates the different molecularweight species. It is concluded, therefore, that during passage over theG-10 column and resulting removal of salt the anti-inflammatory factorformed a large molecular weight aggregate. The degree of aggregationvaried with the salt concentration.

The aggregation property suggests the possibility that a wide spectrumof different molecular weight species can be formed which haveanti-inflammatory biological activity due to the presence of theanti-inflammatory factor. The discovery of this property suggests thepossibility of producing milk anti-inflammatory factors having a widespectrum of different biochemical properties depending on the degree ofaggregation of the final product. For example, formulations havinglonger or shorter biological half lives might be produced by usinglarger or smaller molecular weight aggregates, with molecular weightdistribution being controlled by the salt concentration duringprocessing. The column chromatography method described herein results inthe smallest molecular weight species that has been obtained which hasbiological activity (i.e., peak 2 from the Step 3 G-10 column). Thisobservation also suggests using other methods for forming theaggregates. For example, dilution in water causes the aggregation tooccur. Chemical agents that bind salts, especially calcium, can causethe formation of the aggregate. Having made this discovery, othermethods for forming the aggregate and separating the factor will beobvious to those skilled in the

EXAMPLE 4 Biological Activity Assay

The anti-inflammatory action of purified anti-inflammatory factor wastested on edema that was caused by the injection of a solution ofcarrageenan into the footpads of rats. A lyophilized sample of the milkanti-inflammatory factor preparation was dissolved in the appropriatevehicle and given intraperitoneally to experimental rats. Thecarrageenan was then administered to the rats in an amount of 0.1 ml ofa 1% saline solution in each hind footpad. The footpads were measuredbefore injections were given and 2.5 hours after the injections, using athickness gauge. The results are illustrated in Tables 2 and 3. In theseTables, the abbreviation MAIF refers to the preparation of milkanti-inflammatory factor obtained using the procedures described inExamples 1 and 2 above.

The non-aggregated form of the factor (peak 2 from the G-10 column) fromcontrol and hyperimmune milk caused reduction in inflammation of the ratpaw at doses between 1 mg and 0.25 mg (Table 2). Both the hyper-immunemilk and the regular milk exhibited activity; however, the hyperimmunematerial was more potent. We concluded from this that theanti-inflammatory factor is present in greater concentration in the milkfrom hyperimmune cows.

The second peak from the DEAE column exhibited activity when isolatedfrom either hyperimmune milk or regular milk. The activity issubstantially greater in the hyperimmune milk (Table 3).

The first peak from the G-10 column, which is the aggregated form of thefactor, exhibited activity in rat paw tests (Table 2). However, theaggregated form is not as potent as the nonaggregated form on equalweight basis.

It is concluded from these studies that the anti-inflammatory factoroccurs naturally in cows milk. Hyperimmunization of the cows causeshigher concentration of factor in the milk. The factor is a small,negatively charged molecule that can be separated from the milk by avariety of methods. The factor can form large molecular weightaggregates that do not naturally occur in milk, but form duringprocessing.

                  TABLE 2                                                         ______________________________________                                        Effect of Milk Anti-Inflammatory Factor (MAIF)                                On Reduction of Inflammation in Rats                                                  Foot Pad Measure-                                                             ments (mm)                                                                      Before   After                                                      MAIF Dosage                                                                             Injection                                                                              Injection                                                                              Difference                                                                           % Inflammation                             ______________________________________                                        Prepared from Hyperimmune Milk                                                2.0 mg/rat                                                                              3.43     5.01     1.58   46                                         1.0 mg/rat                                                                              3.49     5.39     1.90   54                                         0.5 mg/rat                                                                              3.42     5.51     2.09   61                                         0.1 mg/rat                                                                              3.43     5.86     2.43   71                                         Control/saline                                                                          3.43     5.82     2.39   70                                         Prepared from Normal Cows Milk                                                2.0 mg/rat                                                                              3.30     5.24     1.94   59                                         1.0 mg/rat                                                                              3.31     5.22     1.91   58                                         0.5 mg/rat                                                                              3.32     5.33     2.01   61                                         0.25 mg/rat                                                                             3.31     5.42     2.11   64                                         ______________________________________                                    

                  TABLE 3                                                         ______________________________________                                        Comparison of Semipurified Fractions of MAIF on                               Reduction of Inflammation in Rats                                             (Prepared from Hyperimmune and Regular Milk)                                          Foot Pad Measurements                                                         (mm)                                                                          Before 2.5 hr. After      %                                                   Injection                                                                            Injection Difference                                                                             Inflammation                                ______________________________________                                        DEAE Column                                                                             3.25     5.04      1.79   55                                        Second Peak                                                                   Hyperimmune                                                                   Milk 2 mg/rat                                                                 DEAE Column                                                                             3.30     5.24      1.94   59                                        Second Peak                                                                   Regular Milk                                                                  2 mg/rat                                                                      G-10 Column                                                                             3.31     4.98      1.67   50                                        First Peak                                                                    2 mg/rat                                                                      Control/Saline                                                                          3.34     5.63      2.29   69                                        ______________________________________                                    

EXAMPLE 5 Chemical Analysis of Anti-inflammatory Factor

Anti-inflammatory factor samples were analyzed chemically. The factor isnot crystalline in structure, as determined by X-ray diffractionstudies. MAIF preparations gave an elemental analysis consistent withcarbohydrate composition. The C, H, O ratios were consistent with apolymeric or oligomeric material with some carbinol groups beingoxidized to carboxyl. The slight excess of calcium equivalents overchloride ions may be accounted for in part as carboxylate salts. Theremainder may be sodium or potassium salts. However, the meltingbehavior, or rather the non-melting behavior, was suggestive of salt-Ekeand/or higher molecular weight compositions. The material in the presentstate of purity apparently contains a variable amount of salts ofcalcium and chloride, probably CaCl₂.

Neither preparation contained a significant amount of nitrogen whichprecludes any peptide component in its composition. Likewise, theabsence of significant nitrogen can rule out the presence of aminosugars and other nitrogen-containing materials such as various complexlipids as the major component (s).

Pyrolytic mass spectra revealed significant traces of 18-carbon fattyacids. This fact, taken together with traces of N and P, suggest thepresence of a complex lipid in the preparation.

Infrared spectroscopy revealed absorptions consistent with carbinol andcarboxylate functionalities. Ultraviolet, visible and fluorescentspectroscopy revealed no significant amount of chromophores beyond thoseindicated by infrared.

The chemical tests are consistent with an oligomeric carbohydrate,wherein the carbonyl function (aldehyde or ketone) is tied up in thesubunit linkages. The oligomeric carbohydrate also contains someside-chain oxidation to carboxylate.

The MAIF preparation is substantially, but not completely pure.

EXAMPLE 6 Rat Paw Edema Tests: Oral Administration

The rat carrageenan footpad assay was used to test the effectiveness ofthe anti-inflammatory factor as an in vivo anti-inflammatory agent.Thirty adult white rats were randomly divided into three groups of tenrats per group. The groups received, in five consecutive dailytreatments, either 10 mg of skim milk powder from hyperimmunizedanimals, 10 mg of skim milk powder from non-immunized animals or notreatment (20 ml water per day only). The powders were orallyadministered in 20 ml of water. On the fifth day the right paw of eachrat was injected with 0.1 ml of 1% carrageenan in saline. This procedureis known to cause acute inflammation (edema). Twenty-four hours afterinjection, the rats were sacrificed, the paws amputated, and the weightsof the left (control) and right (edematous) paws were compared. Theresults of the assay are shown in Table 4 (expressed as weight in grams)and in FIG. 3 (expressed as a percentage of the average weight ofcontrol paws).

                  TABLE 4                                                         ______________________________________                                        Rat Paw Edema Test Results                                                    (Paw wt, g, mean ± sem, n = 10)                                                       Carrageenan Control   Difference                                   Treatment  Paw (wt, g) Paw (wt, g)                                                                             (g)                                          ______________________________________                                        Immune Milk                                                                              1.78 + 0.03 1.71 + 0.02                                                                             0.06 + 0.02                                  Control Milk                                                                             1.88 + 0.06 1.64 + 0.03                                                                             0.24 + 0.05                                  Water      1.86 + 0.03 1.65 + 0.03                                                                             0.22 + 0.02                                  ______________________________________                                    

The inflammatory response to carrageenan injection was markedly reducedin the immune milk treated rats as compared with the nonimmune milk andwater control groups. No evidence of side effects or adverse effects onthe general health of the rats was detected. From these data it can beconcluded that daily consumption of skim milk powder from hyperimmunizedanimals almost completely blocked the inflammatory response induced bycarrageenan injection in the footpad of rats.

EXAMPLE 7 Quantitative Rat Paw Edema Tests

A series of experiments was conducted on the hyperimmune milk fraction.The experiments were designed to confirm the anti-inflammatory activityof the milk anti-inflammatory factor when given intraperitoneally and toestablish a dose response curve, explore alternative routes ofadministration, and investigate dosage regimens which might form thebasis of further investigations.

Peak I from the G-10 column, supplied by Stolle Milk BiologicsInternational, was prepared according to the methods described in PatentNo. 4,956,349. Lactose, obtained from commercial sources, was used asplacebo. Aspirin was used as a positive control Aspirin was dissolved inwater and given orally by gastric garage at the ratio of 200 mg perkilogram, a dose known to be active in the assay. A 2% solution of kappacarrageenan (Sigma C-1263) has been found to produce the mostreproducible results and was thus used in these experiments. The footpadassay was modified by using isotopically labeled human serum albumin(¹²⁵ I-HSA) that localizes in the carrageenan-induced lesion in directproportion to the volume of the exudate. By determining the totalradioactive count in the footpad and comparing this to the counts in aknown volume of plasma from the injected animal, a direct measurement ofedema in microliters of plasma equivalents is obtained. ¹²⁵ I-HSA wasinjected intravenously at a dose of 1.0 microcurie per rat. Female DarkAgouti rats were used. The rats were approximately 12 weeks old, weighedbetween 160 grams and 200 grams, and were obtained from the in-houseinbred colony.

To conduct the carrageenan footpad assay, 0.1 ml of 2% carrageenan wasinjected subcutaneously into each hind foot pad of an anesthetized rat.This injection was followed immediately by injection of 1.0 microcurieof ¹²⁵ I-HSA in 0.5 ml of saline into the tail vein. After four hours,each rat was weighed, blood samples obtained, and the rat euthanized.Both hind feet were then removed and the levels of radioactivity in eachfoot and in the 200 μl plasma standard were measured in an automatedgamma counter. From these measurements the volume of edema in each footwas calculated and expressed in microliters.

Experiment 1: Intraperitoneal Dose Response.

FIG. 4 illustrates the effect of intraperitoneal administration of apurified preparation of MAIF compared to lactose (CON), aspirin, and notreatment (No R_(x)). All treatments (lactose, aspirin, MAIF) were given30 minutes prior to the injection of carrageenan.

Carrageenan injection resulted in edema averaging 250 μl (No R_(x)). Theedema was inhibited by aspirin and all dosages of the MAIF preparationbut was not inhibited by lactose. The intraperitoneal dose-responsecurve obtained with the MAIF preparation, derived by expressing the dataas percentage of average control (no treatment) edema is shown in FIG.5.

Experiment 2: Effects of Various Routes of MAIF Administration.

FIG. 6 illustrates the effect, on footpad edema, of the administrationof lactose and a preparation of purified MAIF orally (ORAL),intramuscularly (IM), subcutaneously (SUB Q), and intravenously (IV).Also shown are a positive control (aspirin) and a nontreated control (NOR_(x)).

The preparations were administered prior to carrageenan challengeaccording to the following schedule: Aspirin: orally, 30 minutes prior;Subcutaneous MAIF: 1 hour prior; Oral MAIF: 24, 16 and 1 hour prior;intramuscular MAIF: 30 minutes prior; intravenous MAIF: at the time ofchallenge (isotope was also injected).

The results indicate that, expressed as the percentage of averagecontrol edema in each separate assay, the anti-inflammatory factor, byall routes of administration, inhibited edema formation. Fortymilligrams of the MAIF preparation given intravenously almost completelyabrogated the inflammatory response to carrageenan. These resultsdemonstrate the anti-inflammatory activity of MAIF and, in view of theresults of Experiment 1 above, suggest that the order of effectivenessfor different routes of administration is IV>IP>IM>SUB Q>ORAL.

Experiment 3: Effect on Edema of Intravenous and Extended OralAdministration: Dose Response.

FIG. 7 shows the effects of IV and oral administration of a purifiedpreparation of anti-inflammatory factor on footpad edema in rats. MAIForal treatment (40 mg per rat per day) was given daily for six days andalso one hour before carrageenan challenge (PO). Intravenous treatments(5, 10, 20 mg) were given at the time of carrageenan challenge (IV).Also shown are a positive control (aspirin) and a negative control (notreatment).

The results shown in FIG. 7 indicate that all three dosages of the MAIFpreparation result in anti-inflammatory activity that exceeds even theactivity of aspirin in the assay, whereas extended oral administrationresults in marked but limited activity.

The study was therefore extended to examine the effects of furtherreduced intravenous dosages of anti-inflammatory factor. Intravenousdosages of lactose placebo were included as a control. The results ofthese studies are shown in FIG. 8. Intravenous dosages of 2.5 and 1 mgof the MAIF preparation (IV) induced anti-inflammatory activity in therange of the activity induced by aspirin. 10 ml of intravenous lactoseplacebo (10 mg PLAC IV) did not induce activity in that range.

An intravenous dose-response curve was derived by combining the resultsof Experiments 2 and 3 and expressing these results as percentageaverage control edema (no treatment) in each separate assay. The curveis shown in FIG. 9.

The conclusions that may be drawn from the quantitative rat paw edematests are as follows: milk fraction peak I from the G-10 column,extracted and purified as described in U.S. Pat. No. 4,956,349,consistently shows anti-inflammatory activity when tested in the rat pawedema model. A dosage of 4 mgs of MAIF preparation per rat givenintravenously at the time of carrageenan injection is sufficient todrastically inhibit edema and was therefore chosen as a standard againstwhich other preparations would be compared in further experiments.

EXAMPLE 8 Anti-Inflammatory Properties of Preparations of HyperimmuneMilk Obtained from Identical Twin Cows

The effect of vaccination on the anti-inflammatory activity of milk wasinvestigated by testing the bioactivity of various milk fractionsobtained from identical twin cows. Based on the extraction methodsdescribed in U.S. Pat. No. 4,956,349, an extraction scheme utilizingultra-filtration was devised. The processing sequence was as follows:##STR1##

Milk samples were prepared from immunized twin cows, non-immunizedcontrol twin cows, and reconstituted skim milk powder previouslyprepared from immunized cows. The sample group consisted of 45 sets ofidentical twin cows. One cow of each twin set was vaccinated bi-weeklywith Stolle S100 mixed bacterin (described in U.S. Pat. No. 4,956,349).The bioactivity of the various fractions was tested by intravenousinjection using the rat carrageenan footpad assay described above.

The hypotheses to be tested were that (a) hyperimmunization wasresponsible for the anti-inflammatory activity described above. (b) MAIFcould be extracted on a commercial scale by ultra-filtration, and (c)dilution of the permeate would cause aggregation of theanti-inflammatory factor, causing it to be retained by the 30,000molecular weight ultra-filtration membrane.

FIG. 10 illustrates the results of a twin herd ultra-filtrationexperiment designed to test the bioactivity of various fractions madefrom the milk of non-vaccinated control twins and from reconstitutedmilk powder from immunized cows. The fractions that were tested are asfollows: Peak I, G-10 column preparation, 4 mls (OHIO MAIF STD); R₂final retentate from non-vaccinated twin (CONTROL TWIN R₂); P₂ finalpermeate from the reconstituted milk powder (RECON S100 P₂); dialyzed R₂final retentate from non-vaccinated twin (CON DIALYZED R₂); dialyzedfinal retentate from the reconstituted milk powder (S100 DIALYZED R₂).

No anti-inflammatory activity could be detected ha the R₂ finalretentate fraction prepared from nonimmunized cows, even after dialysis.No anti-inflammatory activity was detected in the final permeate P₂fraction prepared from the reconstituted milk powder. The reconstitutedmilk powder retentate R₂ fraction, following dialysis, exhibitedanti-inflammatory activity ha the range of the activity of the MAIFstandard.

FIG. 11 illustrates the results of twin herd ultra-filtrationexperiments designed to test the bioactivity of various milk fractionsmade from vaccinated and nonvaccinated twin cows and from reconstitutedmilk powder from immunized cows. The fractions that were tested are asfollows: Peak I, G-10 column preparation, 4 ml (OHIO MAIF STD); dialyzedfinal retentate R₂ from non-vaccinated twins (CON DIALYZED R₂); finalretentate R₂ from the reconstituted milk powder (RECON S100 R₂); thefinal retentate R₂ from vaccinated twins (IMMUNE TWIN R₂); firstretentate R1 from the reconstituted milk powder, diluted for: 1 (S100DILUTED R1).

Little anti-inflammatory activity was detected in the dialyzed retentateR₂ from non-vaccinated control twins or in the non-dialyzed retentate R₂from the vaccinated twins. Some activity is detectable by scattergram.R₂ retentate prepared without dialysis from reconstituted Stolle milkpowder from immunized cows was strongly anti-inflammatory. However, thepreparation made by dilution of the reconstituted milk beforeultrafiltration rather than dilution of whey made from the milk was onlymarginally active. This result indicates that anti-inflammatory activityis more efficiently extracted from the whey fraction.

FIG. 12 illustrates the results of twin herd ultrafiltration experimentsdesigned to test the bioactivity of dialyzed retentate from vaccinatedtwin cows. The fractions tested are as follows: Peak I, G-10 columnpreparation (OHIO MAIF STD); dialyzed final retentate R₂ from vaccinatedtwins (IMM DIALYZED R₂); dialyzed final retentate from the G-10preparation (DIALYZED OHIO MAIF). The results show thatanti-inflammatory activity was present in the R₂ fraction from theimmunized twin after dialysis. Dialyzed MAIF was more active in theassay than the nondialyzed MAIF standard. This result suggests thatdialysis is an effective means of further concentrating the milk factorresponsible for anti-inflammatory activity.

The results presented in FIGS. 10-12 above support the followingconclusions: (1) anti-inflammatory activity can be extracted fromreconstituted milk from immunized cows by ultrafiltration of the dilutedpermeate. (2) anti-inflammatory activity was not demonstrated in theabove-preparations that were made from the milk of non-immunized cows.(3) anti-inflammatory activity was demonstrated in the final retentateR₂ after ultrafiltration of diluted permeate prepared from the milk ofimmunized cows, but dialysis was necessary in order to demonstrate theactivity.

EXAMPLE 9 Stability of MAIF, Heating, and Proteinase Treatment of MAIF

The previous evidence that the milk anti-inflammatory factor waschemically not a protein or a peptide was based largely on chemicalanalyses that consistently showed an almost complete absence ofnitrogen. For further characterization of the anti-inflammatory factor,several preparations were tested in the rat paw edema assay, using 4 mgsof peak I, G-10 column preparation, intravenously as the standard. Thefollowing treatments were done: proteinase (pronase) treatment for sixhours; six hours no proteinase treatment control; untreated positivecontrol; heating at 100° C. for 30 minutes.

The results of this assay are illustrated in FIG. 13. The conclusionsderived from this study were that the anti-inflammatory activity is notdue to a protein or peptide and that the anti-inflammatory factor is notinactivated by boiling. The effectiveness of pronase treatment wasverified by the finding that parallel pronase treatment completelydenatured milk protein.

EXAMPLE 10 Anti-Inflammatory Activity of Further Purified MAIF and WheyProtein Concentrate from Immunized Cows

Retentate and permeate from ultrafiltration using an Amicon YM5 membranewere tested for biological activity using intravenous administration inthe rat paw edema assay. In this process, the MAIF in peak I of the G-10column, prepared according to U.S. Pat. No. 4,956,349, was furtherpurified by ultrafiltration on an Amicon YM5 membrane. This membraneretains molecules of 5000 molecular weight or greater. Whey proteinconcentrates (WPCs) were also prepared from milk from immunized animalsand filtered through the YM5 membrane. The following samples were testedin the assay using 4 mg peak I, G-10 column preparation, intravenouslyas the standard: permeate from Amicon YM5 ultrafiltration; retentatefrom Amicon YM5 ultrafiltration; WPC from immunized cows, 30 mgs perrat; WPC from commercial production (non-immunized cows), 30 mg per rat.

The results of this assay are illustrated in FIG. 14. It is clear fromthese results that all of the activity is in the retentate whichcomprised approximately 0.5% of the total weight of the fraction appliedto the YM5 filter. The reduction of edema seen in this experiment wasachieved following administration of 20-25 micrograms of material.

Regarding the activity of WPC, WPC made from hyperimmunized animalsclearly showed anti-inflammatory activity as expected. Interestingly,WPC made from non-immunized animals also showed anti-inflammatoryactivity. The presence of anti-inflammatory activity in the milk ofnonimmunized cows is not surprising since it must be a naturalsubstance. Its detection reflects the sensitivity of the bioassay.

EXAMPLE 11 Continuous Monitoring of Carrageenan Induced Footpad Edema

It was established that 4 mg of MAIF preparation given intravenously atthe time of carrageenan injection reduced the accumulation of edema inthe footpad by between 40% and 50%. Although these results providedevidence that the material contained an anti-inflammatory moiety, therewas little indication of the site of action or pharmacological profileof MAIF. In order to obtain such data it was necessary to establish amethod that allowed the continuous monitoring of footpad edemathroughout the response to carrageenan. This was achieved by holding therat foot in a demounted Gamma radiation detector. The procedure requiredanimals to be anesthetized for up to four hours and, as anesthetics areknown to suppress the inflammatory response, it was first necessary todetermine the effect of anesthetics on the carrageenan-induced edema.Five gents commonly used to induce anesthesia in rats were thereforeevaluated; these were ether, chloral hydrate, Innovar-vet, nembutal andurethane. The results are shown in FIG. 15.

It was clear from these results that ether was the anesthetic of choicewhen the inflammatory response was to be evaluated by this technique.The shape of the curve obtained when ether was used indicated a biphasicresponse. To delineate the response in more detail a further experimentwas carried out in which the volume of edema was measured at 12 timepoints over a 5 hour period. The results confirmed a biphasic response.The early response occurred between 0 and 1 hour after challenge andlate phase response between 1.5 and 2 hours (FIG. 16).

The two phases, which have also been observed by other investigators,have been termed the non-phagocytic inflammatory response (NPIR) and thephagocytic inflammatory response (PIR), respectively.

The NPIR is initiated, in response to injury, by soluble mediators suchas histamine and bradykinin while the PIR depends on the participationof neutrophils. The protocol, therefore, was to administer MAIF andmonitor the accumulation of edema continuously in an effort to determinewhether the anti-inflammatory properties of the gent were a result of aneffect on the early non-cellular (NP1R) or the later cellular (PIR)phase. 5 mg or 40 mg of MAIF preparation per rat were administeredintravenously at the time of carrageenan challenge and the accumulationof edema monitored at regular intervals over a four hour period. Neitherdose affected the accumulation of edema during either phase (FIG. 17).

This result was surprising as many previous analyses, in which theeffect of purified preparations of MAIF on carrageenan induced edema 4hours after challenge was determined, had demonstrated considerableanti-inflammatory activity in the fractions. It was likely, therefore,that the continuous exposure to ether suppressed or inactivated theactive anti-inflammatory component of MAIF in vivo.

Previous studies indicated that short term exposure to ether did notaffect the activity of the anti-inflammatory factor. Therefore, anexperiment was done in which the effect of MAIF on progressive edemaaccumulation was determined at only four time points, 0, 1, 3 and 4hours, thus limiting the exposure of the animals to ether. The 1 hourtime point was chosen to assess the affect on the early non-phagocyticinflammatory response while the 3 and 4 hour measurements were selectedto quantify the effect on the later phagocytic inflammatory response. Inthis experiment the MAIF preparation administered at 40 mg resulted in areduction in the accumulation of edema during the secondary,phagocytic-cell mediated phase, but had no significant effect on theprimary, soluble mediator driven phase (FIG. 18).

The following conclusions can be drawn from this series of experiments.

1. Ether is the preferred anesthetic for use in experiments where theinflammatory response to carrageenan is to be monitored continuously.

2. Continuous ether anesthesia inhibits the in vivo anti-inflammatoryactivity of anti-inflammatory factor in the carrageenan footpad assay.

3. MAIF ameliorates inflammation by inhibiting the late, phagocytic-cellmediated phase of the inflammatory response to carrageenan.

EXAMPLE 12 Time Course of the Effect of MAIF on Carrageenan InducedFootpad Edema

A further series of experiments were carried out in which the agent wasadministered at selected time points before or after the injection ofcarrageenan rather than at the time of challenge. The purpose of thestudy was to provide information on

(a) the most effective time for administration of MAIF in relation tothe inflammatory stimulus.

(b) the biological half life of the anti-inflammatory moiety.

(c) the points in the development inflammatory response affected byMAIF.

The study was carried out in three parts. A preparation of MAIF wasadministered intravenously at a dose of 4 mg/rat at one of 11 timepoints, ranging from 150 minutes before, to 150 minutes after injectionof carrageenan. Results of this experiment are shown in FIG. 19 andTable 5.

                                      TABLE 5                                     __________________________________________________________________________           Time of A                                                                            Mean foot      Inhibition of                                           in relation                                                                          volume of                                                                            Mean foot                                                                             edema by MAIF                                           to challenge                                                                         control groups                                                                       volume of MAIF                                                                        (% of control                                    Experiment                                                                           (min)  (μl ± SD)                                                                      groups (μl ± SD)                                                                volume ± SD)                                  __________________________________________________________________________    3      -150   311 ± 65                                                                          246 ± 52                                                                           79 ± 17                                       2      -90    304 ± 71                                                                          211 ± 33                                                                           73 ± 11                                       2      -60    304 ± 71                                                                          186 ± 34                                                                           61 ± 11                                       1      -30    391 ± 63                                                                          261 ± 49                                                                           67 ± 13                                       3      -15    311 ± 65                                                                          152 ± 41                                                                           49 ± 13                                       1, 2, 3                                                                               0     336 ± 78                                                                          184 ± 42                                                                           55 ± 13                                       3       15    311 ± 65                                                                          218 ± 30                                                                           70 ± 10                                       1       30    391 ± 63                                                                          218 ± 30                                                                           56 ± 8                                        2       60    304 ± 71                                                                          212 ± 40                                                                           69 ± 13                                       2       90    304 ± 71                                                                          216 ± 37                                                                           70 ± 12                                       3      150    311 ± 65                                                                          261 ± 42                                                                           84 ± 14                                       __________________________________________________________________________

A significant inhibition of edema was observed at all time pointsstudied; however, the level of inhibition was less at the outer extremes(±150 min). An interesting cyclic response to MAIF administration wasseen in those groups treated closer to the point of challenge. The factthat MAIF was more effective when given 30 minutes after challenge thanwhen given 15 minutes after challenge supports the concept that thesecondary, phagocytic-cell mediated, phase of the response is inhibitedby the agent. The preparation of MAIF strongly inhibited the response tocarrageenan when administered 15 minutes before or at the time ofchallenge. It is apparent, furthermore, that the agent has a relativelylong half life in the serum (1-2h) and its effectiveness is related tothe time of challenge and the dynamic nature of the inflammatoryresponse.

It is thus surmised that the anti-inflammatory effect is due to aneffect on inflammatory cells, likely the neutrophils.

EXAMPLE 13 Effect of MAIF on the Reverse Passive Arthus Reaction

The possibility that the anti-inflammatory factor might affectneutrophil involvement was investigated by evaluating the ability of thematerial to modulate the reverse passive Arthus reaction (RPA). Thisimmune complex-induced response is primarily neutrophil mediated andagents which affect the development of the reaction do so via an effecton these cells. To induce the RPA, rats were injected intradermally withrabbit antibody to ovalbumin and intravenously with native ovalbumin.Ovalbumin/ovalbumin-antibody immune complexes form in and around thedermal blood vessel wails, host neutrophils bind to the Fc portion ofthe antibody and an intense inflammatory reaction is initiated. Itshould be noted that, although the response is initiated byimmune-complexes, it takes place independently of the host's immunesystem.

Three parameters are used to quantify the RPA. These are, (1)edema--measured using the accumulation of ¹²⁵ I-HSA, (2)hemorrhage-assessed by in vivo pre-labelling RBC's with ⁵⁹ Fe and (3)neutrophil accumulation--measured by determining tissue levels of theneutrophils specific enzyme myeloperoxidase (MPO). These assays areknown to those of ordinary skill in the art.

Eighteen rats were divided into three groups of six. Rabbitanti-ovalbumin (40 μl) was injected intradermally at four sites on theback of each animal and 2 mg of ovalbumin injected intravenouslyimmediately afterwards. One group of animals received no other treatmentand served as controls. The second group were injected intravenouslywith 20 mg of a lactose preparation, while the final group were injectedintravenously with 20 mg of a purified preparation of MAIF. Both lactoseand MAIF preparation were administered with the ovalbumin. The severityof the reaction was assessed 3.5 hours after challenge. When the MAIFpreparation was administered intravenously at a dose of 20 mg/rat priorto the initiation of the RPA response, there was a highly significantinhibition of the three parameters used to measure the response (Table6, FIG. 20). The lactose control material also caused a modest andmarginally significant suppression of neutrophil accumulation andhemorrhage. This indicates that there is a small amount ofanti-inflammatory activity in normal milk.

                  TABLE 6                                                         ______________________________________                                                Neutrophil                                                                    accumulation:           Haemorrhage:                                  Group   Units of MPO  μl of Edema                                                                          μl of RBC                                  ______________________________________                                        Control  0.30 ± .157                                                                             107 ± 29                                                                             4.8 ± 3.1                                  Lactose  0.214 ± .176**                                                                          104 ± 23                                                                             3.0 ± 1.5**                                MAIF    0.056 ± .013                                                                              60 ± 27*                                                                            1.5 ± 1.7*                                 ______________________________________                                         *= p < 0.01                                                                   **= p < 0.05                                                             

As the neutrophil is the primary mediator of the RPA, these resultsprovided additional evidence that MAIF was capable of inhibiting theinflammatory response via an effect on neutrophil function.

EXAMPLE 14 Effect of MAIF on Neutrophil Migration

In order to participate effectively in an inflammatory response,neutrophils must tint migrate from the vasculature to the site ofinflammation. To determine whether anti-inflammatory factor interferedwith neutrophil migration, a model of inflammation employing thesubcutaneous implantation of sterile polyurethane sponges was used. Thesponges are removed at intervals after implantation and by weighing thesponges and then extracting and counting the cells in the infiltrate,both the fluid and cellular phase of the response can be quantified.Twenty four hours after implantation >95% of the cells found in thesponge are neutrophils.

Two experiments have been carried out. In the first, animals weretreated with either 5, 10, 20, or 40 mg of a purified MAIF preparationat the time of sponge implantation. Sponges were removed 24 hours afterimplantation. Each group consisted of between 5 and 8 rats and twosponges were implanted in each animal. The results are shown in FIG. 21.

Twenty or 40 mg of MAIF preparation, administered intravenously at thetime of sponge implantation, had a marked effect on the ability ofinflammatory cells to migrate. A less marked, but equally significant,inhibition of fluid accumulation was also seen. The two lower doses ofMAIF had no demonstrable effect in this model of inflammation.

A second experiment, designed to delineate the temporal relationshipbetween the inflammatory challenge (sponge implantation) and MAIFadministration, was carried out. In this study, 20 mg of MAIFpreparation were administered intravenously 30, 60 or 120 minutes aftersponge implantation. A fourth, control, group was left untreated. Therewere five animals in each group. Two sponges were implanted in eachanimal and these were removed after 24 hours. The results areillustrated in FIG. 22. Included on this graph are results obtained froma sample group of rats that received 20 mg of the MAIF preparation atthe time of implantation (see FIG. 21).

Results from the time-course of the effect of MAIF oncarrageenan-induced footpad edema show MAIF to be comparativelyineffective when administered 60 minutes or later after challenge. It isnoteworthy that while 20 mg of the MAIF preparation was required tosuppress the inflammation associated with the sponge implantation, 4 mgwas sufficient to inhibit the carrageenan-induced edema. Withoutintending to be held to this interpretation, this disparity may berelated to the different level of provocation presented to the host bythe two stimuli. The sponge implant is a relatively benign stimuluswhich induces a slow inflammatory response and the bulk of the cellsaccumulate between 8 and 16 hours after implantation (FIG. 23). On theother hand the subcutaneous injection of carrageenan is a very strongstimulant which induces a correspondingly strong response over arelatively short period (FIG. 16).

EXAMPLE 15 Alternative Method of purifying the Anti-Inflammatory Factorfrom Milk (Preparation "AIF")

The following Example describes a method for purifying theanti-inflammatory factor from milk in its lowest molecular weight,unaggregated form. The preparation resulting from the purification stepsdescribed herein has been given the designation "AIF" in order todistinguish it from the preparation obtained using the proceduredescribed in Example 2. In the present Example and in the Example whichfollows (i.e. Example 16) the active factor within the preparations isreferred to simply as the "anti-inflammatory factor". All of thepurification steps were performed so as to minimize possiblecontamination with bacteria or pyrogens. Sterile water was used toprepare solutions and all glassware was depyrogenated.

Step 1: <10,00 Molecular Weight ("MW") Ultrafiltration

Fresh S100 immune skim milk (see Example 1 for a description ofprocedures used in obtaining the immune milk) was pumped through a10,000 MW cutoff ultrafiltration membrane (Filtron) at a pressure of 30psi. The permeate was collected in depyrogenated bottles maintained onice. Permeates were sterile filtered and refrigerated until use. The<10,000 MW permeate contains the milk anti-inflammatory factor as wellas low molecular weight peptides, oligosaccharides and a large amount oflactose. Anti-inflammatory activity in the permeate occurs in a lowmolecular weight, unaggregated form.

Step 2: DEAE-Sepharose Chromatography

Initial fractionation of anti-inflammatory activity was performed onDEAE-Sepharose. A 5×50 cm column containing one liter of DEAE-Sepharosewas equilibrated with permeate buffer. Permeate buffer is a sterile,endotoxin-free solution containing the diffusible ions found in bulkmilk in the appropriate concentrations. Permeate buffer contains CaCl₂,MgCl₂, NaCl, NaCitrate and NaH₂ PO₄. Typically, approximately eightliters of <10,000 MW permeate were pumped onto the DEAE-Sepharose columnat a flow rate of about 500 ml per hour. Column eluate was monitored at280 nm. The column was washed with distilled water until the 280absorbance returned to baseline (about 6 to 8 liters of distilled waterwere typically required). Anti-inflammatory activity was bound to thecolumn and was eluted with about 4 liters of 0.5M ammonium acetate inwater, pH 7.4. The eluate was lyophilized to dryness and weighed. Theweight of recovered material obtained from eight liters of permeate wastypically between 15 and 20 grams. Since ammonium acetate is completelyvolitized during lyophilization, the residual weight represents theweight of bound material. Anti-inflammatory activity was assayed in themouse neutrophil migration inhibition assay.

Step 3: H-40 Chromatography

The material eluted from the DEAE-Sepharose column was furtherfractionated on a sizing column in order to separate the factorresponsible for anti-inflammatory activity from other low molecularweight components. Eight grams of the DEAE sample was dissolved in 50 mlof distilled water and applied to a 2.5×150 cm column containing 736 mlof Toyopearl HW-40 (Rohm and Haas) equilibrated in water. The column wasdeveloped in distilled water at a flow rate of 40 ml per hour and eluatewas monitored at 280 nm. Fractions were collected and assayed foranti-inflammatory activity in the mouse neutrophil migration inhibitionassay. Fractions evidencing activity and minimal absorbance at 280 nmwere pooled and lyophilized. Approximately 80 mg of material containinganti-inflammatory activity was recovered from eight liters of permeate.

Step 4: AffiGel 601 Chromatography

The final purification step involved affinity chromatography of theactive factor in a column packed with a boronate-derivatizedpolyacrylamide based medium (AffiGel 601, Bio-Rad) which has an affinityfor coplanar adjacent cis hydroxyl groups. Forty mg of low molecularweight HW40 derived material was equilibrated in 10 ml of 0.25M ammoniumacetate, pH 7.0, and applied to the AffiGel column which had also beenequilibrated in 0.25 ammonium acetate. Eluate was monitored at 280 nm.The column was washed with 400 ml of 0.25 M ammonium acetate at a flowrate of 50 ml per hour until the 280 nm absorbance decreased tobackground. The AffiGel column was then eluted with 1600 ml of 0.1Mformic acid, pH 2.8. The eluate was tested for activity in the mouseneutrophil migration inhibition assay and lyophilized to dryness.Approximately 8 to 10 mg of bound material containing anti-inflammatoryactivity was recovered from 8 liters of permeate.

The preparation obtained by this method is given the designation "AIF".The preparation was substantially purified with respect to theanti-inflammatory factor but is not homogeneous. The preparationexhibits anti-inflammatory activity in the mouse neutrophil migrationinhibition assay, in the rat paw edema assay, in the rat ear swellingassay and blocks neutrophil binding to rat mesentery venule endothelium(visualized by intravital microscopy). Based upon comparative analysesin the mouse neutrophil migration inhibition assay, AIF is approximately55,000 fold more purified than the original skim milk <10,000 MWpermeate.

EXAMPLE 16 Effect of Preparations of Anti-inflammatory Factor on theAdhesion of Neutrophils to Endothelial Cells and on the Emigration ofNeutrophils from the Vasculature

The effect of the anti-inflammatory factor on the adhesion ofneutrophils to endothelial cells and on the emigration of neutrophilsfrom the vasculature was tested. Two different preparations ofanti-inflammatory factor were used. One preparation was made using thepurification procedure described in Example 2. For the purposes of thepresent Example, this preparation is referred to simply as "MAIF". Theother preparation of anti-inflammatory factor was made using thepurification procedure described in Example 15 and is referred to bothin that Example and in the present Example as "AIF". It is to beunderstood that both MAIF and AIF contain within them theanti-inflammatory factor at different states of purity.

Chemicals:

Human serum albumin, trypsin, platelet-activating factor (PAF), phorbolmyristate acetate (PMA), propidium iodide, and Histopaque were obtainedfrom Sigma Chemical Co., St. Louis, Mo. Human Neutrophil elastase waspurchased from Calbiochem. A murine anti-human CD18 monoclonal antibody(IgG₁ -subclass; FITC conjugate) and a murine anti-keyhole limpethemocyanin (IgG₁ -subclass; FITC conjugate), used as a negative controlantibody, were purchased from Becton Dickinson Systems Inc., MountainView, Calif. Simply Cellular™ Microbeads were purchased from FlowCytometry Standards Corp., Research Triangle Park, N.C. Other reagentswere the best grade commercially available and were used without furtherpurification.

In Vivo Methods:

Intravital microscopy experimentation. Twenty-four male Wistar rats(180-250 g) were maintained on a purified laboratory diet and fasted for24 hr prior to surgery. The animals were initially anesthetized withpentobarbital (12 mg/100g body weight). A right carotid artery andjugular vein were cannulated to measure systemic arterial pressure(Statham P23A pressure transducer and a Grass physiologic recorder) anddrug administration respectively. A midline abdominal incision was madeand the animals were placed in a supine position. A segment of themid-jejunum was exteriorized through the abdominal incision and allexposed tissue was covered with saline soaked gauze to minimize tissuedehydration. The mesentery was carefully placed over an optically clearviewing pedestal that allowed for transillumination of a 2 cm² segmentof tissue. The temperature of the pedestal was maintained at 37° C. witha constant temperature circulator (Fisher Scientific, model 80). Rectaland mesenteric temperatures were monitored using an electrothermometer.The mesentery was suffused with warmed bicarbonate-buffered saline (pH7.4). An intravital microscope (Nikon Optiphot-2, Japan) with an X25objective lens (Leitz Wetzlar L25/0.35, Germany) and X10 eyepiece wasused to observe the mesenteric microcirculation. A video camera mountedon the microscope projected the image onto a color monitor and theimages were recorded for playback analysis using a video cassetterecorder. Single unbranched venules with diameters ranging between 25and 40 μm were selected for study. Venular diameter was measured on lineusing a video caliper. The number of adherent and emigrated neutrophilswas determined off-line during playback of videotaped images. Aneutrophil was considered adherent to venular endothelium if it remainedstationary for 30 seconds or more. Rolling neutrophils were defined asthose white blood cells that moved at a velocity less than that oferythrocytes in the same vessel. Leukocyte rolling velocity wasdetermined by the time required for a leukocyte to traverse a givendistance along the length of the venule.

Experimental protocol. After all hemodynamic parameters were in steadystate, images from the mesentery were recorded for 5 minutes. Themesentery was then superfused for 60 minutes with 100 nM PAF in thepresence of either 40 or 5 mg/rat of the MAIF preparation (iv.).Measurements of aforementioned parameters were again performed at 30 and60 min of PAF superfusion. In two experimental groups, the mesentericpreparations were again exposed to PAF as described above but, at 30minutes, they received either 40 or 5 mg/rat of the MAIF preparation. Inthree additional experiments, the AIF preparation was given either as apretreatment or as a post-treatment.

In Vitro Methods

Isolation of Neutrophils. Neutrophils from healthy donors were purifiedby dextran sedimentation followed by hypotonic lysis and Histopaquecentrifugation. Except for the dextran sedimentation step, which wasperformed at room temperature, the cells were kept at 4° C. throughoutthe isolation procedure. Cell preparations contained 95% neutrophils andgreater than 99% of these were viable as determined using Trypan Blue.After isolation, neutrophils were resuspended at a final concentrationof 2×10⁶ cells/ml in phosphate buffered saline (PBS). Aliquots of cellswere then incubated at 37° C. for 20 minutes with varying concentrationsof either the MAIF or the AIF preparation. After washing, neutrophilswere incubated in the dark at 4° C. for 30 minutes with saturatingconcentrations of fluorescein-conjugated murine anti-human CD18, humanCD11b, IGG coated microbeads (Simply Cellular™ microbeads) or the murinenegative control antibody.

Immunofluorescence Staining and FACS Analysis. Direct immunofluorescenceas a measure of CD18 surface expression was determined by analysis on aFACScan (Becton Dickinson Systems Inc., Mountain View, Calif.) using thechannel number (log scale) representing the mean fluorescence intensityof 10,000 cells. The logarithmic channel numbers were convened to linearvalues using methods well-known in the art. The specific meanfluorescence intensity for cells stained by CD18 antibodies wascalculated after subtracting the mean fluorescence intensity of thecells exposed to the negative control antibody. Non-viable cells werescreened out using propidium iodide.

Superoxide Assay. Superoxide production from isolated neutrophils wasmeasured following PMA and N-formyl-Met-Leu-Phe ("fMLP") stimulation inthe presence of various concentrations of MAIF. The reduction ofcytochrome C by activated neutrophils was measured using aspectrophotometer (Hitachi U2000) at 550 nm. Briefly, sample was addedto two cuvettes and one cuvette was used as a reference. The lattercontained superoxide dismutase (superoxide scavenger). Neutrophils wereallowed to equilibrate at 37° C. for 5 min in the presence of variousconcentrations of MAIF and the cells were then stimulated with eitherPMA or fMLP. Superoxide production was measured for 3 min.

Protease Release. ¹²⁵ I-labelled albumin was coated onto wells andallowed to dry overnight. Unbound albumin was washed and thenPMA-stimulated neutrophils were incubated within the wells for one hourin the presence or absence of various concentrations of MAIF. Freeradioactivity within the supernatant of the wells was divided by totalradioactivity within each well to assess the level of proteolysis.

Results:

Results are summarized in FIGS. 24-30 and Tables 7-9. FIG. 24demonstrates that PAF superfusion increased neutrophil adhesion topostcapillary venules approximately 6-fold over a 60 min period. 40mg/rat of the MAIF preparation reduced the PAF-induced neutrophiladhesion by more than 90% at 30 minutes and by more than 80% at 60minutes. Interestingly, MAIF pretreatment seemed to also reduce thenumber of adherent neutrophils prior to exposure of PAF. The lowerconcentration of MAIF (5 mg per rat) was less effective, reducingleukocyte adhesion by 50% at 60 min. The AIF preparation at aconcentration of 0.01 mg per rat was found to reduce leukocyte adhesionby about 50% at 60 min. At a tenfold higher concentration of AIF, a verylarge increase in leukocyte adhesion was observed (data not shown). Theadhesion was so dramatic that the videotape could not be analyzed. FIG.25 shows the effect of MAIF and AIF on neutrophil emigration. MAIF at aconcentration of 40 mg per rat and 5 mg per rat and AIF at aconcentration of 0.01 mg per rat were found to completely prevent theincrease in neutrophil emigration with time of PAF exposure. Neutrophilflux did not appear to change significantly in the MAIF treated groupcompared with the untreated group (FIG. 26). When AIF was given, weinitially observed more neutrophils robing than usual, however thenumber decreased with time.

In a second series of experiments, the various anti-inflammatory agentswere administered after neutrophils were already adherent (FIG. 27). Inthis series of experiments, leukocyte adhesion was reversed by an MAIFdose of 40 mg/rat but not by a dose of 5 mg/rat. AIF at a dose of 0.01mg per rat reversed neutrophil adhesion by approximately 25%. To furtherassess the effect of the higher concentration of MAIF (40 mg/rat), thenumber of adherent neutrophils at the start of the recording procedureand the number of new neutrophils that adhered over 5 min at each periodwere examined. FIG. 27a demonstrates that there were fewer neutrophilsadherent following 10 min of MAIF administration indicating that theanti-inflammatory factor had actually "peeled off" adherent neutrophils.Moreover, FIG. 27b clearly demonstrates that MAIF blocked newneutrophil-endothelial cell adhesions. The speed with which neutrophilsrobed along the length of venules did not change between groups or withtime with the exception that AIF may have increased neutrophil rollingvelocity (FIG. 28). This effect was rather interesting in light of thefact that red blood cell velocity remained unchanged (FIG. 29). Theresults suggest that a simple increase in hydrodynamic forces cannotexplain the increase in neutrophil rolling velocity. Neutrophil fluxalso was unaffected by MAIF but was again reduced by AIF (FIG. 30).

In vitro data indicates that the anti-inflammatory factor does notinterfere with the activation of neutrophils per se. The superoxideradical scavenger, superoxide dismutase completely blocked cytochrome creduction by PMA and fMLP-stimulated neutrophils, suggesting that thisis a superoxide-mediated process. MAIF at extremely high concentrationsonly minimally affected cytochrome c reduction, suggesting that MAIFdoes not directly scavenge superoxide (Table 7). Protease release wasnot affected by MAIF (data not shown).

It was found that the binding of anti-CD18 monoclonal antibody could bereduced with MAIF or AIF (Table 8). This did not occur with the CD11bantibody. Binding of CD18 antibody to IgG coated microbeads was also notaffected by the MAIF or AIF preparations suggesting that theanti-inflammatory factor was not affecting the ability of the anti-CD18monoclonal antibody to bind to substrate but was, more likely, actingupon the Egand, CD18. The same pattern was observed with stimulatedneutrophils (Table 9). It should be noted that the binding to CD18varied between days because different cells were used each day.Therefore, a direct comparison of the results in Table 8 with those inTable 9 cannot be made.

                  TABLE 7                                                         ______________________________________                                        Effect of MAIF on Superoxide Secretion by Cells                                          PMA-Stimulated fMLP-Stimulated                                                Superoxide Production                                                                        Superoxide Production                               MAIF       (nmole/min/10.sup.7 cells)                                                                   (nmole/min/10.sup.7 cells)                          ______________________________________                                        0.0   mg/ml    153            55                                              0.1   mg/ml    145            50                                              1.0   mg/ml    143            40                                              5.0   mg/ml    140            32                                              10.0  mg/ml    127            --                                              ______________________________________                                    

                  TABLE 8                                                         ______________________________________                                        Effect of Anti-Inflammatory Factor on the                                     Availability of CD18 and CD11 Cell Surface Antigens                                      Mean Channel   Mean Channel                                        Unstimulated                                                                             Fluorescence   Fluorescence                                        Neutrophils                                                                              Anti-CD18 Antibody                                                                           Anti-CD11 Antibody                                  ______________________________________                                        Neutrophils alone                                                                        314.24         1594.57                                             +0.1 mg/ml MAIF                                                                          234.26         1553.74                                             +1.0 mg/ml MAIF                                                                          262.78         1796.00                                             +0.1 μg/ml AIF                                                                        248.28         1577.04                                             +1.0 μg/ml AIF                                                                        188.93         1554.61                                             Beads + Anti-CD18 Antibody                                                                   60.03                                                          +1 mg/ml MAIF  88.61                                                          +1 μg/ml AIF                                                                              84.99                                                          ______________________________________                                    

                  TABLE 9                                                         ______________________________________                                        Effect of MAIF on the Availability of CD18 Cell Surface Antigens              on Stimulated and Unstimulated Neutrophils                                                  Mean Channel Fluorescence                                       ______________________________________                                        Unstimulated Neutrophils                                                                      236.95                                                        + MAIF 1 mg/ml  216.08                                                        + MAIF 5 mg/ml  251.51                                                        Stimulated Neutrophils                                                                        266.69                                                        + MAIF 1 mg/ml  158.68                                                        + MAIF 5 mg/ml  171.96                                                        ______________________________________                                    

Discussion:

The data in the above Example suggests that the anti-inflammatory factorprevents neutrophil adhesion and emigration in venules in adose-dependent manner. More importantly however, the anti-inflammatoryfactor could, within a brief period (10 min), reverse neutrophiladhesion to these vessels. The only other agents that cause adherentneutrophils to release their hold on the endothelium with suchefficiency are monoclonal antibodies directed against the CD11/CD18glycoprotein complex on the neutrophil. MAIF did not appear to have anyeffect on blood flow through the individual vessels or on systemic bloodpressure, suggesting that hemodynamic factors such as shear stress couldnot account for the reversal of leukocyte adhesion. Although leukocyterolling appears to be a prerequisite for leukocyte adhesion, MAIF didnot effect leukocyte rolling velocity or leukocyte flux. The latterresult suggests that the anti-inflammatory factor did not affect thenumber of neutrophils that rolled through the vessel and therefore, thatthe reduction in adherent leukocytes was not a result of fewerleukocytes interacting with the endothelium. The fact that leukocyterolling velocity as well as leukocyte flux remained unchanged suggeststhat adhesion molecules on neutrophils and endothelium responsible forleukocyte rolling (1L-selectin, P-selectin) were not affected by theanti-inflammatory factor in the MAIF preparation..

It has been reported that the leukocyte may regulate its own adhesion byreleasing superoxide as well as proteases. It was therefore conceivablethat the lack of leukocyte adhesion in the presence of MAIF and AIF wasdue to the ability of these preparations to block the release superoxideor proteases. This possibility is untenable in light of the fact thatMAIF had little effect on superoxide or protease release and did notinteract with released proteases or scavenge released superoxide.Moreover, the MAIF did not appear to affect neutrophil viability asassessed with propidium iodide making a direct cytotoxic effect of theanti-inflammatory factor on neutrophils unlikely.

For a neutrophil to adhere and emigrate it must have an intact CD11/CD18 glycoprotein complex. Immunoneutralization of the adhesion complexcompletely impairs the ability of the neutrophil to permanently adhereto the endothelium and emigrate into the surrounding tissue. Sinceneutrophil adhesion and emigration is a rate limiting step in the tissueinjury associated with a number of inflammatory conditions, an agentthat interferes with these processes would also likely block theinflammatory response. In the present study, both MAIF and AIFdramatically reversed neutrophil adhesion and blocked neutrophilemigration induced by PAF. Because of the similarity between AIF-, MAIF-and anti-CD18 monoclonal antibody induced reversal of neutrophiladhesion, it seemed possible that the anti-inflammatory factor withinAIF and MAIF exerted its effect by directly interacting with the CD18glycoprotein complex. The in vitro data presented above supports thisview, in that both AIF and MAIF blocked the ability of an anti-CD18antibody to bind to the CD18 glycoprotein complex. In contrast, neitherAIf nor MAIF affected the binding of CD11b to its respective monoclonalantibody. Finally, the AIF and MAIf preparations did not interfere withthe ability of the anti-CD18 monoclonal antibody to bind to IgG-coatedmicrobeads. Therefore, it can be concluded that the anti-inflammatoryfactor interacts with the CD18 complex directly and prevents CD18 frombinding to various ligands, including endothelial cell adhesionmolecules.

EXAMPLE 17 Effect of MAIF on Circulating Leucocytes

Several pharmacological agents can inhibit neutrophil migration. Whilesome, such as cyclophosphamide, are cytoreductive and act by inhibitinghemopoiesis in the bone marrow, other agents, such as steroids and thenon-steroidal anti-inflammatory drugs, have specific sites of action anddo not result in leucocytosis. It was important therefore to determinethe effect of the anti-inflammatory factor on circulating white bloodcell numbers and ratios.

Two experiments were done. In the first, the MAIF preparation wasadministered intravenously at a dose of 40 mg/rat to one group of 6animals and a control group was injected with saline. Blood samples wereobtained at baseline, 1, 4, and 24 hours after treatment. The resultsare summarized in FIG. 31.

MAIF administration resulted in an increase in circulating neutrophilnumbers, maximal at 4 hours, and a corresponding decrease in the numberof peripheral blood lymphocytes. A further dose-response study wascarried out in which a group of rats were injected intravenously withsaline, 5, 10 or 20 mg of the MAIF preparation. Blood from each rat hadbeen taken 7 days previously to provide baseline values and was takenagain 4 hours after the injection of MAIF. The results are shown in FIG.32. Included on the graph are the results obtained from the sample taken4 hours after the administration of 40 mg the MAIF preparation (see FIG.31).

All doses of MAIF resulted in an increase in the number of circulatingneutrophils and a decrease in the number of lymphocytes. While theeffect on lymphocytes was linearly related to dose, the increase inneutrophil numbers was in the form of a curve, the greatest effect beingobserved in those animals given 10 mg.

These results support the concept that the anti-inflammatory factormodulates inflammation by affecting the adhesion of neutrophils toendothelial cells.

Data were also obtained pertaining to the effect of three othercell-targeted, anti-inflammatory/immunomodulatory agents on circulatingleucocytes in the rat. The steroidal drug, methylprednisolone, causes achange in the lymphocyte/neutrophil ratio analogous to that seen withMAIF. The temporal relationship between drug administration and effectis somewhat different. The anti-rejection/anti-inflammatory agentcyclosporin A also causes an increase in the number of circulatingneutrophils but lymphocyte numbers are either increased or not affecteddepending on the dose. In contrast, the cytotoxic drug cyclophosphamidedepletes both circulating lymphocytes and neutrophils. The effects ofthe anti-inflammatory factor would appear to closely parallel the actionof methyl-prednisolone.

EXAMPLE 18 Effect of the Anti-inflammatory Factor on Lymphocyte Function

The ability of the anti-inflammatory factor to induce a reversibledecrease in the number of circulating lymphocytes (Example 17) promptedfurther investigation of the effect of the factor on lymphocytefunction. Graft versus Host (GvH) and Host versus Graft (HvG) analyseswere used to determine the effect of the factor on T lymphocytefunction.

In the HvG analysis, parental Dark Agouti rats ("DA") were injected i.v.with 20 mg of the MAIF preparation 48, 24 and 3 hours before lymphocytesfrom their F1 hybrid offspring (DA×Hooded Oxford rats) were injectedinto their footpads. Thus, the effect of the anti-inflammatory factor onthe ability of T lymphocytes from an intact host (DA) to respond to theforeign histocompatibility antigens of the F1 lymphocytes was measured.The protocol produced a highly significant reduction (30%) in theresponse as evidenced by a decrease in popliteal lymph node weight (FIG.33A).

In the GvH reaction parental (DA) lymphocytes were obtained from MAIFtreated parental rats (DA) and injected into the footpads of their F1(DA×Hooded Oxford) offspring. This assay measured the in vivoresponsiveness of T lymphocytes removed from the host under evaluation,i.e. from MAIF treated rats. The MAIF regimen had no effect on the GvHresponse (FIG. 33B).

During the preceding experiments, an apparent increase in the number ofsplenic lymphocytes in MAIF treated animals was noted. Furtherexperiments showed a significant increase in both spleen weight and inspleen cell numbers (FIGS. 33C and 33D). The increase in spleen cellnumbers was approximately equal to the decrease in the number ofcirculating cells reported previously.

Finally, the effect of the anti-inflammatory factor on the ability ofisolated splenic lymphocytes to respond to the mitogen concanavalin Awas determined. Administration of the MAIF preparation was found toalmost totally abrogate the mitogenic response of cultured lymphocytesto this lectin (FIG. 33E).

EXAMPLE 19 Suppression of Infection Induced Inflammation by theAnti-inflammatory Factor

Experiments have been carried out to determine whether changes in serumlevels of acute phase reactants (APRs) could be used to quantify theanti-inflammatory activity of the anti-inflammatory factor. The APRs area group of proteins which are synthesized in response to an inflammatorystimulus. One of these, alpha 2 macroglobulin, is common to both man andrats and methodology for measuring this inflammatory component isavailable. Two intravenous injections of MAIF preparation (0 and 24hours) did not reduce the peak response (48 hours) of alpha 2macroglobulin. This result indicates that the factor does not affect thelater inflammatory response.

EXAMPLE 20 In Vitro and in vivo Evaluation of Milk DerivedAnti-Inflammatory Factor (Bovine Mammary Macrophage Assay, InfectionModels in Mice)

Incubation of bovine mammary macrophages with the hyperimmune milkfraction did not detectably enhance the degree of phagocytosis but didincrease the ability of macrophages to kill phagocytosed Staphylococcusaureus. Mice injected intraperitoneally with 10 mg of the MAIFpreparation per kilogram demonstrated increased resistance tointraperitoneal challenge with lethal Staphylococcus aureus.

In an intra-mammary Staphylococcus aureus mastitis challenge model, MAIFinjected mice also showed significantly less mammary inflammation andinvolution and increased clearance of the infectious organism.Quantitative histological analysis of mammary tissue from MAIF treatedmice showed significantly more lumen, less interalveolar connectivetissue, and less leukocytic infiltration compared to control mice.Mammary glands of treated mice also contained fewer colony forming unitsthan control mice. The anti-inflammatory appears to exert its effect onthe non-specific defense system by a modulation of leukocyte function.

EXAMPLE 21 Effect of the Anti-Inflammatory Factor on the Pathogenesis ofExperimental Infection

The most common inflammagens encountered by man are microbial and it isimportant to determine the effect of any agent which modulates hostdefenses against infection. The tissue damage which accompanies manyinfectious diseases is in fact caused by the host response to infectionrather than by the invading organism. While the ability to modulate theinflammatory response to infection could be a useful clinical technique,it must be recognized that inhibition of the host response duringinfection can be disadvantageous. This is especially true in the case ofneutrophil inhibition. Studies with gents which curb the participationof neutrophils in the early stages of infection have demonstrated that,while inflammation and tissue damage may be initially suppressed, theincreased bacterial load that occurs as a result of the reduced cellularresponse eventual leads to an exacerbation of tissue damage. Thus, it isessential to evaluate the potential of the milk anti-inflammatory factorto modulate infection in order to (1) determine if the agent can reduceinfection-induced tissue damage and (2) to assess whether any observedsuppression of the host response is accompanied by an increase in theseverity of infection.

The effect of the anti-inflammatory factor on edema formation followingthe intradermal injection of E. coli 075 was determined. Two groups of 8animals were used. One group was untreated and served as controls whileindividuals in the second group were injected intravenously with 40 mgof the MAIF preparation in 0.5 ml saline. Immediately after theadministration of MAIF, 100 μl of an overnight culture of E. coli 075was injected intradermally at two skin sites on the shaved back of therat, followed by the intradermal injection of 100 μl of saline at twofurther sites. To allow estimation of edema volume in the infected skin,0.1 μCi of ¹²⁵ I-HSA was injected intravenously at the time ofchallenge. Six hours later the animals were anaesthetized, a bloodsample obtained, the skin on the back removed and the infected andsaline injected sites punched out. The volume of edema was calculated byrelating tissue counts to plasma counts as described. To obtain thevolume of edema which accumulates as a result of the presence of E. colithe edema/plasma volume of the saline-injected sites was subtracted. Theresults are shown in FIG. 34.

MAIF administration resulted in a 48% inhibition of edema formation.This experiment established that the anti-inflammatory factor couldmodulate the local inflammatory response to infection.

In order to study the relationships between anti-inflammatory factoradministration, bacterial replication, the accumulation of fluid andinflammatory cell infiltration, an alternative model of infection wasemployed. Polyurethane sponges, prepared and implanted as previouslydescribed, were infected with a quantitated sample of E. coli 075 at thetime of implantation. The sponges were removed at timed intervals,weighed to determine the volume of the fluid exudate, and then squeezedin media to free the bacteria and cells from the sponge. Bacterial andcell numbers were estimated using techniques known to those skilled inthe art. The following experiment was carried out using this model.Ninety animals were divided into two groups of 45. One of these groupswas untreated and served as controls. The second group were injectedintravenously with 40 mg of the MAIF preparation. The sponges were thenimplanted subcutaneously and, at the time of implantation, each spongewas inoculated with 10⁵ E. coli 075. Groups of 6-8 animals were killedat intervals thereafter and the bacteriological status and the size ofthe inflammatory infiltrate in the sponges determined. The results areillustrated in FIGS. 35-37.

The rate of bacterial replication was much greater in MAIF treatedanimals than in the controls and there was a 10, 1000 and 10,000 folddifference in bacterial numbers at 4, 8 and 16 hours respectively.Thereafter, bacterial numbers declined, although there was still a largedifference at 96 hours (FIG. 35).

The early response to infection is the critical determinant in theoutcome of an infectious episode. In this experiment the cellularinfiltrate at 2, 4 and 8 h in those animals given MAIF was 27%, 35% and46% of the control infiltrate respectively (FIG. 36B). The cells whichaccumulate in the first 24 h after challenge are >90% neutrophils andthe suppression of this cellular component during this phase may accountfor the rapid increase in bacterial numbers. The accumulation of fluidat 2 hours was not affected by the administration of MAIF, but wassignificantly less 4, 8 and 16 hours following challenge. This isconsistent with the previous finding that the anti-inflammatory factordid not suppress the primary, non-cellular phase of edema formation inthe carrageenan footpad model. In previous studies, using theimmunomodulatory agents cyclosporin A and methylprednisolone, a similarassociation between the suppression of the acute cellular inflammatoryinfiltrate and the promotion of bacterial replication was shown.However, in these experiments, the increased bacterial load promoted ahost response between 24 and 48 hours post challenge in which there wasa massive influx of neutrophils. When tissue was involved, the enhancedinflammatory response resulted in a marked exacerbation of tissue damageand scar formation. Interestingly, although administration of MAIFsuppressed the early inflammatory response and was associated with a10,000 fold increase in bacterial numbers there was no massive influx ofneutrophils 24-48 hours post challenge.

EXAMPLE 22 Effect of the Anti-Inflammatory Factor on ExperimentalPyelonephritis

An agent which can suppress inflammation in infection without resultingin a sequela of enhanced tissue damage would have considerablepotential. A clinically relevant model of infectious disease couldprovide an experimental basis for establishing such potential.

Pyelonephritis is an infectious disease which demonstrates localinflammation, tissue destruction and scar formation as cardinalhistological features. A well characterized model of the disease isavailable, which reproduces the central pathological features of thedisease in man. Pyelonephritis is induced in the rat by the directinoculation of the surgically exposed kidney with a predetermined numberof E. coli 075. Following challenge, bacterial numbers increase rapidlyand reach a peak 3 to 4 days later. In normal animals the level ofinfection declines over the following 5 or 6 days and reaches a plateauat about 10 days post challenge. By 21 days the lesions have resolvedand present as focal areas of indented scar tissue. To assess the effectof the anti-inflammatory factor on this model of infection,pyelonephritis was induced in both kidneys of twenty-six animals. Onehalf of these animals were treated with the MAIF preparationintravenously at a dose of 40 mg/rat at the time of challenge and again48 hours later. Seven animals from each group were killed 4 days afterinduction of pyelonephritis and the two remaining groups of six animalsat 21 days. Kidneys were removed aseptically and weighed to determinethe relative volume of the fluid exudate. The extent of the surfacelesion size was estimated by direct visualization and the kidneyhomogenized to allow the enumeration of bacterial numbers. The resultsare shown in FIG. 38.

Four days after challenge the inflammatory response, as evidenced by theinhibition of fluid accumulation and the size of the lesions on thesurface of the kidney, was suppressed by the administration of MAIF. Aspreviously observed in the studies involving infected,subcutaneously-implanted sponges, the early suppression of inflammationresulted in a logarithmic increase in the number of bacteria inMAIF-treated animals. By 21 days there was no difference in thepathology of disease as measured by kidney weight, bacterial numbers orrenal surface lesions size. Thus, while suppression of the earlyinflammatory response with MAIF did not result in a reduction in tissuedestruction in the chronic (21 day) phase of pyelonephritis, neither didit promote the development of pathological lesions as otheranti-inflammatory and immunomodulatory agents have done.

EXAMPLE 23 Summary of Experimental Data

A method was developed which allowed the accumulation of edema in thecarrageenan injected footpad to be monitored continuously.

The early, non-phagocytic, phase of the inflammatory response was notaffected by anti-inflammatory factor, whereas the later,cellular-driven, phase of the reaction was significantly inhibited.Further experiments, in which MAIF was administered at intervals beforeor after the injection of carrageenan, provided additional evidence thatMAIF exerted its anti-inflammatory effect by modulating the secondary,neutrophil-mediated, inflammatory response.

The anti-inflammatory factor was shown to have a half-life of 1-2 hoursfollowing i.v. injection and development of inflammation could besuppressed when the factor was administered 30 minutes after challenge.This result is relevant to the potential therapeutic use of theanti-inflammatory factor.

The neutrophil is the principal cell involved in the acute inflammatoryresponse. During the Arthus reaction, a >80% reduction in neutrophilaccumulation was observed following MAIF administration which, in turn,was associated with a highly significant inhibition of the secondarycharacteristics of the inflammatory reaction, namely edema andhemorrhage. This result further implicated neutrophils as a target inMAIF-induced suppression of inflammation.

One of the key steps in the development of inflammation is the migrationof neutrophils from the vasculature to the tissue. The intravenousadministration of the anti-inflammatory factor was shown to result inprofound and dose dependent inhibition of neutrophil migration. When theeffect of the anti-inflammatory factor on peripheral blood leukocyteswas investigated, a marked increase in the number of circulatingneutrophils was observed, accompanied by a corresponding decrease in thenumber of lymphocytes. This effect was also dose-dependent, but in thecase of the increase in neutrophil numbers, was not linear.

The administration of the milk anti-inflammatory factor was found toboth block the adhesion of neutrophils to the endothelium and to promotethe dissociation of hose neutrophils which were adherent at the time ofadministration. This effect is probably the result of the ability of theanti-inflammatory factor to block the interaction between cell surfaceCD18 antigens and other molecules. The inhibition of CD18 binding by thefactor appears to be specific in that the factor prevented the bindingof anti-CD18 monoclonal antibody to cells but did not similarly preventthe binding of anti-CD11b monoclonal antibody.

The blocking of intermolecular interactions involving the CD18 cellsurface antigen may also account for the observation that the factor wasable to inhibit the ability of host lymphocytes to respond to foreignhistocompatibility antigens. In other experiments, the anti-inflammatoryfactor was found to block the concanavalin-induced mitogenic response inlymphocytes.

Finally, the factor significantly suppressed the early cellular responseto infection, an effect which resulted in a logarithmic increase inbacterial numbers in a model of subcutaneous infection. Thisexacerbation of infection did not result in a rebound of theinflammatory response, as seen with other agents which suppress acuteinflammation in infection. A second experiment using a clinicallyrelevant model of infection, pyelonephritis, also demonstrated asuppressive effect on inflammation which was associated with an increasein bacterial numbers. Again no rebound effect was observed and there wasno difference in the degree of tissue damage which occurred in the MAIFtreated and control groups.

The following conclusions can be drawn from this series of experiments:

1. Anti-inflammatory factor, administered i.v., suppresses thesecondary, neutrophil-mediated, phase of the carrageenan inducedinflammatory response.

2. When evaluated in the carrageenan footpad assay the anti-inflammatoryfactor has a biological half-life of 1-2 hours and is effective evenwhen administered after inflammation is induced. Subsequent experimentsindicate that the effective half-life is dependent on both the dose andinflammatory stimulus employed.

3. Anti-inflammatory factor inhibits neutrophil emigration in vivo.

4. Anti-inflammatory factor administration results in an increase in thenumber of circulating neutrophils and a corresponding decrease inlymphocyte numbers.

5. Anti-inflammatory factor suppresses host defenses against infection,probably via an effect on neutrophil emigration.

6. Anti-inflammatory factor blocks interactions between cell surfaceCD18 antigens and other molecules.

7. Anti-inflammatory factor blocks the adhesion of neutrophils to theendothelium.

8. Anti-inflammatory factor promotes the dissociation of adherentneutrophils from the endothelium.

9. Anti-inflammatory factor blocks the ability of host lymphocytes torespond to foreign histocompatibility antigens.

10. Anti-inflammatory factor blocks the mitogenic response oflymphocytes.

The experimental data obtained in these studies demonstrate clearly thatmilk anti-inflammatory factor has a marked effect on both neutrophilsand lymphocytes. The effects observed may be the result of a directeffect of anti-inflammatory factor on cells per se, or the result of thesuppression (or stimulation) of some other cellular or soluble mediatorwhich indirectly alters the biological activities of cells. It is alsowidely accepted that most pharmacological agents have multiple actionsand it is possible that the anti-inflammatory factor will be found toaffect a number of other, as yet unidentified, biological processes.

EXAMPLE 24 Method of Obtaining Highly Purifed MAIF

Standard methods for preparation of MAIF similar to those describedearlier in this application produce a certain degree of purification ofMAIF from skimmed milk. The following protocol, however, results in amore highly purified preparation of MAIF than previously obtainable.

Materials. Chemicals were all reagent grade. Water used for large-scalepreparative procedures was sterile water for injection.

Standardized MAIF Preparation. In order to compare the MAIF activitiesin different batches of milk from hyperimmune cows or from control cows,a standardized procedure involving ultrafiltration and ion exchangechromatography was adopted to prepare a partially purified, highlyactive sample. A convenient volume (3 to 100 liters) of fresh, coldskimmed milk was subjected to ultrafiltration through the Minisette unit(Omega membrane) at 30 psi until the volume of filtrate collected wasequal to 2/3 of the starting volume of milk. A convenient volume ofstandardized, <10,000 daltons (<10K daltons) permeate (250 ml, 500 ml,etc.) was applied to a DEAE column that was 26.7% of the permeatevolume. The column was washed with a volume of deionized water 2 timesthat of the permeate and eluted with a volume of 0.15M NaCL equal to58.3% of the permeate volume. The MAIF activity in standardized DEAEpreparations was determined in the neutrophil migration inhibition assay(see below) and was defined as the percent inhibition of neutrophilmigration induced by a 3 mg/120-150 gm. rat dose.

Highly Purified MAIF Preparation

Ultrafiltration. Ninety liters of fresh, cold (4° C.), non-pasteurizedskimmed milk from hyperimmunized cows was pumped through a 25° C.in-line warming bath and then passed through a Minisette (Filtron)ultrafiltration cassette containing an Omega (model #OS010C01) membranewith a molecular weight cut-off of <10K daltons. Inlet pressure wasmaintained at 30 psi. The resulting permeate (<10K permeate) wascollected on ice until a volume of sixty liters was accumulated. The<10K permeate was used immediately for further purification by ionexchange chromatography or was frozen and stored at -20° C. or waslyophilized.

Ion Exchange Chromatography. Sixty liters of cold fresh <10K permeatewas applied directly to a 37 cm×15 cm column (Pharmacia, Model KS370/15) containing 16 liters of diethyl-aminoethyl (DEAE)-Sepharose FastFlow ion exchange resin (Pharmacia #17-0709-05) at a flow rate of 4.8liters/hr using a low pressure peristaltic pump. The column effluent wasmonitored at 280 and 254 nm with a Pharmacia Dual Path UV monitor (Model19-24217-01). The loaded column was washed with 120 liters of steriledeionized water to elute unbound material until the 280 nm absorbance ofthe effluent returned to baseline. Bound components were eluted with 35liters of 0.15M NaCl. The column eluate was lyophilized and stored inamber glass vials in the dark. MAIF activity in column eluates wasdetermined in the neutrophil migration inhibition assay (see below).

Size Exclusion Chromatography. Partially purified DEAE preparations ofMAIF were separated on a preparative TSK G2500PW (21.5×600 mm) sizeexclusion HPLC chromatography column (TosoHaas, Montgomeryville, Pa.)equilibrated with 0.15M NH₄ OAc on a Hewlett-Packard Model 1090 HPLCwith an automatic injector and a diode array detector. LyophilizedDEAE-MAIF (100 mg) was dissolved in 0.25 ml of 0.15M NH₄ OAc andinjected onto the column. The column was eluted with 0.15M NH₄ OAc at aflow-rate of 5 ml/min. The effluent was monitored at 220 and 280 nm,however, diode array data was stored for all wavelengths between 190 and600 nm. Fractions (9 ml) were collected on an LKB ultrorac fractioncollector. To prepare large quantities of MAIF, multiple samples of 100mg each were separated on the TSK column and collected into the same setof tubes. Tubes containing the active factor were pooled, lyophilizedand weighed.

Organic Partition Extraction. MAIF was selectively isolated from boundsalts by an organic partition extraction method in which thesemipurified TSK-MAIF sample was dissolved in distilled water,alkalized, extracted with n-hexane to remove neutral lipids, acidifiedand then extracted with ethyl acetate. 50 to 100 mg of TSK-MAIF wasdissolved in 2 ml deionized water in a tared extraction vessel. The pHof the solution was adjusted to 8.0 with 4 drops of 0.02N NH₄ OH. Twomilliliters of n-hexane were added and the mixture was shakenvigorously. The upper phase consisting of hexane was removed, dried andweighed. The remaining aqueous solution was acidified to pH 3.5 with 100drops of citric acid. Five milliliters of ethyl acetate was added andthe mixture was shaken vigorously and centrifuged at 1000 rpm toseparate layers. The ethyl acetate layer was transferred to a ratedvessel. The extraction was repeated with a second 5 ml volume of ethylacetate. The ethyl acetate layers were combined, dried under nitrogenand weighed.

Weak Anion Exchange Chromatography. An aminopropyl Supelcosil (Supelco)HPLC column was used to separate weakly ionic negatively charged MAIFcomponents freed of bound salts by the ethyl acetate extractionprocedure. The amino-bonded column, which acts as a weak anion exchangerwith aqueous buffers, was equilibrated with 78% ACN/H₂ O. A 50 mgTSK-MAIF sample was dissolved in 25% ACN/3 mM NH₄ OAc and applied to thecolumn. The column was developed with a three-part elution systemconsisting of a 78% ACN/H₂ O to 69% ACN/3 mM NH₄ OAc for 0-15 minfollowed by a 69% ACN/3 mM NH₄ OAc to 65% ACN/3 mM NH₄ OAc from 15-30min at a flow rate of 1 ml/min. The column eluate was monitored at 220and 280 nm. Selected fractions were tested for MAIF activity in theneutrophil migration inhibition assay (see below).

Neutrophil Migration Inhibition Assay. MAIF anti-inflammatory activitywas determined in a pleural neutrophil migration inhibition assay.Female, white laboratory rats weighing 120 to 150 g were injectedintrapleurally with 1.0 ml of 1% kappa carrageenin in PBS to induceneutrophil migration into the pleural cavity. Immediately, the rats wereinjected intraperitoneally with 0.5 ml doses of MAIF samples in PBS. PBSwas used as a control. After four hours, the rats were sacrificed,pleural exudates were collected, and the emigrated neutrophils werecounted.

RESULTS

Standardization of Preparative Methods. Reproducible methods involvingultrafiltration and DEAE column procedures and analysis of comparablesamples in the rat neutrophil migration inhibition assay wereestablished for the production of highly active MAIF preparations. Theadoption of standardized purification methods permitted quantitation ofMAIF activity in specific milk products and in large-scale productionbatches. To evaluate the standardized method, MAIF was prepared fromhyperimmune milk, from control milk obtained from non-immunized cows andfrom a commercial powdered milk and was tested blind in the neutrophilmigration inhibition assay. MAIF samples were tested at doses of 0.5,1.5, 3, 5, and 8 mg/120-150 gm rats for their ability to inhibit themigration of neutrophils to inflammatory sites (FIG. 39). MAIF fromhyperimmune milk produced a maximum neutrophil inhibition of more that70% at the 3 mg dose. Fresh control milk and commercial control milkeach produced only 18% inhibition at the 3 mg dose. The activity ofstandardized MAIF prepared from hyperimmune milk was compared withsialic acid and orotic acid, known components of milk believed to haveanti-inflammatory activity (FIG. 40) and with two anti-inflammatorydrugs (FIG. 41). Neither the sialic acid nor the orotic acid exhibitedany inhibitory activity on the migration of rat neutrophils in the dosestested. Indomethacin exhibited 35% inhibitory activity at a dose of 0.5mg/120-150 gm. rat, however, the inhibition dropped to 30% at 3mg/120-150 gm. rat and declined steadily at higher doses. Aspirin, atrat doses equivalent to human doses, showed less than 10% inhibition andreached the high inhibition range only at doses nearly 100 times thehuman equivalent rat dose.

MAIF Purification by Preparative HPLC. The use of DEAE ion exchangechromatography to prepare MAIF resulted in a 25-fold purification of theanti-inflammatory activity with respect to the (10K permeate. Analysisof the composition of the DEAE-derived MAIF by size exclusion HPLC (FIG.42) indicated the presence of two major components at 17 and 25 minutesand six or more minor components which eluted between 30 and 60 minutes.Analysis of pooled fractions in the neutrophil migration inhibitionassay demonstrated that the major concentration of MAIF activityoccurred in the 25 minute peak. These results indicated that apreparative size separation step would permit the preparation of a morehighly purified preparation of MAIF. Attempts to achieve comparableresults on a preparative liquid chromatography medium did not provideadequate separations. A preparative HPLC TSK2500PW column, eluted with0.15M NH₄ OAc, provided excellent separation of the 25 minute MAIF peak.DEAE-MAIF was separated on the HPLC column in 100 mg runs and the pooled25 minute peak was lyophilized. The 0.15M NH₄ OAc elution buffer wasselected because all buffer salts would be completely removable fromisolated MAIF samples during lyophilization. However, when repeated 100mg runs of DEAE-MAIF were fractionated by preparative HPLC, recoveredweights of the 25 minute peak exhibited only a 10% reduction in weight.Elemental analysis of the MAIF peak indicated that more than 40% of theweight could be attributed to salt. Several salts, including sodiumchloride, sodium acetate and magnesium chloride, were tested todetermine their elution position on the TSK 2500PW column. Each salteluted between 35 and 40 minutes. This result indicated that salt werespecifically bound by the MAIF compound eluting at 25 minutes.

Elimination of Contaminating Salt. In order to free the MAIF compoundfrom bound salts, an organic partition extraction method was used inwhich the HPLC purified MAIF preparation was alkalized to exposenegatively charged groups and extracted with hexane to remove hexanesoluble contaminants. The aqueous phase was then acidified to protenatenegative charges, extracted into ethyl acetate and weighed. Theextracted residue exhibited a reduction in weight of more than 96%.Analysis of the dried ethyl acetate fraction in the neutrophil migrationinhibition assay demonstrated strong MAIF activity and required as muchas a 10,000 fold reduced dose of the highly purified MAIF to obtain thesame level of neutrophil migration inhibition as MAIF obtained prior tothe organic partition extraction (FIG. 43). Analysis of the MAIFcompound on an aminopropyl weak anion exchange column before and afterextraction demonstrated a significant shift in elution position of theremaining components and loss of a should in the profile (FIG. 44).These results indicated that the partition extraction method resulted inthe removal of the contaminating bound salts and permits the preparationof a more highly purified preparation of MAIF than previouslyobtainable.

The standardized MAIF method produces a highly active, partiallypurified form of MAIF in quantities that are sufficient for compositionand structural studies to be conducted. MAIF produced by this methodexhibits high activity when compared to MAIF from control milk or topostulated or known anti-inflammatory drugs. Use of preparative TSK HPLCto fractionate MAIF is effective in producing a more purified MAIF,however, salts remain bound to the MAIF compound in an unexpected way.Organic partition extraction of the MAIF compound into ethyl acetatepermits the removal of the contaminating salts, thus allowing largescale preparation of highly purified MAIF.

All references cited herein are fully incorporated by reference intothis dislosure.

Having now generally described this invention, it will become readilyapparent to those skilled in the art that many changes and modificationscan be made thereto without affecting the spirit or scope thereof. Suchchanges and modifications are also considered aspects of the invention.

What is claimed is:
 1. A method for purifying an anti-inflammatoryfactor from skimmed milk comprising:(i) ultrafiltering said milk througha filter with a molecular weight cut-off of 10,000 daltons; (ii)collecting a <10,000 dalton permeate from step (i); (iii) applying saidpermeate to a first ion exchange column and washing said column withdeionized water; (iv) collecting the eluate from step (iii); (v)separating the eluate from step (iv) on a preparative size exclusionHPLC chromatography column and collecting the eluate; and (vi)performing organic partition extraction of the eluate obtained from saidpreparative size exclusion HPLC chromatography and obtaining the aqueousextract from said extraction.
 2. The method of claim 1 furthercomprising weak anion exchange chromatography of said aqueous extract.3. The method of claim 2, wherein the resin in said first ion exchangecolumn is diethyl-aminoethyl (DEAE)-Sepharose fast flow ion exchangeresin.
 4. The method of claim 3, wherein said preparative size exclusionHPLC column is a TSK G2500PW size exclusion column.
 5. The method ofclaim 4 wherein said organic partition extraction further comprises:(i)extracting with hexane and NH₄ OH; (ii) reextracting the aqueous phasefrom step (i) with ethyl acetate; and (iii) collecting said ethylacetate extract.
 6. The method of claim 5, further comprising:(i)applying said ethyl acetate extract to a weak anion exchange column; and(ii) developing said column with a three part elution system.
 7. Ananti-inflammatory factor in highly purified form produced by a processcomprising:(i) ultrafiltering said milk through a filter with amolecular weight cut-off of 10,000 daltons; (ii) collecting a <10,000daltons permeate from step (i); (iii) applying said permeate to an ionexchange column, and washing said column with deionized water andcollecting an eluate; (iv) separating the eluate from step (iii) on apreparative size exclusion HPLC chromatography column; (v) extractingsamples from step (iv) with hexane and NH₄ OH; (vi) reextracting theaqueous phase from step (v) with ethyl acetate and obtaining an aqueousphase; and (vii) collecting the eluate from said HPLC chromatographycolumn.
 8. An anti-inflammatory factor in substantially or highlypurified form produced by any one of the methods of claims 1 or 2-6. 9.The method of any one of claims 1 or 2-6, wherein the ultrafiltrationstep begins with about 90 to about 225 liters of skimmed milk and theion exchange step begins with about 60 to about 150 liters of permeate.10. The method of claim 9 wherein the HPLC size exclusion chromatographyis performed with 100 mg of milk anti-inflammatory factor from a DEAEion-exchange step.
 11. A method for inhibiting neutrophil migrationcomprising administering to an animal the milk anti-inflammatory factorobtained by the method of any one of claims 1, 2-6, 9 or 10 at a dosesufficient to inhibit said neutrophil migration.
 12. A method forinhibiting the inflammatory response comprising administering to ananimal the milk anti-inflammatory factor obtained by the method of anyone of claims 1, 2-6, 9 or 10 at a dose sufficient to inhibit saidinflammatory response.
 13. A method for inhibiting neutrophils fromadhering to endothelial cells in a mammal, wherein said method comprisesadministering to said mammal the milk anti-inflammatory factor obtainedby the method of any one of claims 1, 2-6, 9 or 10 at a dose sufficientto inhibit said adherence.
 14. A method for detaching neutrophils whichhave adhered to endothelial cells in a mammal, wherein said methodcomprises administering to said mammal the milk anti-inflammatory factorobtained by the method of any one of claims 1, 2-6, 9 or 10 at a dosesufficient to cause said detachment.
 15. The method of claim 14 whereinsaid neutrophils have adhered to said endothelial cells in response toplatelet activating factor.
 16. A method for inhibiting the interactionbetween CD18 cell-surface antigens present in a mammal and moleculeswhich are capable of binding to said antigens, wherein said methodcomprises administering to said mammal the milk anti-inflammatory factorobtained by the method of any one of claims 1, 2-6, 9 or 10 at a dosesufficient to inhibit said interaction.
 17. A method for inhibiting theemigration of leukocytes from the venous system of a mammal wherein saidmethod comprises administering to said mammal the milk anti-inflammatoryfactor obtained by the method of any one of claims 1, 2-6, 9 or 10 at adose sufficient to inhibit said emigration.
 18. The method of claim 17,wherein said leukocytes are neutrophil.
 19. A method for suppressing themitogenic response of lymphocytes in a host mammal to foreign antigens,wherein said method comprises administering to said mammal the milkanti-inflammatory factor obtained by the method of any one of claims 1,2-6, 9 or 10 at a dose sufficient to suppress the mitogenic response.20. The method of claim 19, wherein said antigens are on the cellsurface.
 21. The method of claim 20 wherein said cells are leukocytes.22. The method of claim 21, wherein said cells are lymphocytes.